Chromobacterium Subtsugae Genome

ABSTRACT

Disclosed herein is the nucleotide sequence of the Chromobacterium subtsugae genome. Also provided are the nucleotide sequences of open reading frames in the C subtsugae genome (i.e., C. subtsugae genes). In addition, the amino acid sequences of proteins encoded by the C. subtsugae genome are provided. Nucleic acids, vectors and polypeptides comprising the aforementioned sequences are also provided. Homologues, functional fragments and conservative variants of the aforementioned sequences are also provided. Compositions having pesticidal, bioremedial and plant growth-promoting activities comprising C. subtsugae genes and proteins, and methods for the use of these compositions, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application and claims the benefit ofU.S. application of U.S. patent application Ser. No. 16/177,656 filedNov. 1, 2018, which is a Divisional application of U.S. patentapplication Ser. No. 15/510,369 filed on Mar. 10, 2017 now U.S. Pat. No.10,160,976, which is a 371 Application of and claims the benefit ofInternational Application Serial No. PCT US/2015046045 filed on Aug. 20,2015, which claims the benefit of U.S. Provisional Patent ApplicationNo. 62/049,016 filed on Sep. 11, 2014. The content of all of which areincorporated by reference herein in their entirety.

INCORPORATION OF SEQUENCE LISTING

This instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy is namedMBI-203-0006-US-PR1_ST25.txt and is 24,039,565 bytes in size.

INCORPORATION BY REFERENCE

Incorporated herein by reference is the table submitted herewithelectronically via EFS-Web in ASCII format under the file nameMBI-203_Table1forfilingAllGenesTabDelimited.txt. This table containssequences of open reading frames and sequences encoding Chromobacteriumsubstugae polypeptides and proteins. The sequences in said table intheir entirety comprise a substantial portion of the Chromobacteriumsubtsugae genome.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

FIELD

The present disclosure is in the field of biopesticides; in particularbacterial pesticides, their genes and their gene products.

BACKGROUND

Chromobacterium subtsugae

In 2000, a purple-pigmented bacterium (PRAA4-1) was isolated from forestsoil in Maryland (Martin et al., 2004). In initial screens, thisbacterium was found to be toxic to Colorado potato beetle and otherinsect pests (Martin et al., 2007a). Additional work with the isolaterevealed activity gainst mites, grubs, diverse beetle species, aphidsand plant parasitic nematodes, among other plant pests (Martin et al.,2007b, US Patent Application Publication No. 2012/0100236 A1).

Proteases and Insect Control

Proteases have the ability to target and destroy essential proteins andtissues of insects. Plants have naturally evolved to express proteasesto protect against insects. Insect predators also produce protease intheir venom, which contributes to mortality. Proteases have beenidentified as important insecticidal agents for control of insects inagriculture.

Proteases with insecticidal activity fall into three general categories:cysteine proteases, metalloproteases and serine proteases. Proteases ofthese classes target the midgut, cuticle and hemocoel. The peritrophicmatrix of the midgut is an ideal target for insect control because itlines and protects the midgut epithelium from food particles, digestiveenzymes and pathogens; in addition to acting as a biochemical barrier(Hegedus at al., 2009). Enhancins are zinc metalloproteases expressed bybaculoviruses that facilitate nucleopolyhedrovirus infections inlepidopterans (Lepore et al., 1996). These proteases promote theinfection of lepidopteran larvae by digesting the invertebrateintestinal mucin protein of the peritrophic matrix, which in turnpromotes infection of the midgut epithelium (Wang and Granados, 1997).Homologs of enhancin genes found in baculovirus have been identified inthe genomes of Yersinia pestis, Bacillus anthracis, Bacillusthuringiensis and Bacillus cereus (Galloway et al., 2005; Hajaij-Ellouzeet al., 2006).

Plant cysteine proteases also demonstrate activity against lepidopteranlarvae. Cysteine proteases in the latex of the papaya and wild fig treesare essential in the defense against various lepidopteran larvae.Toxicity to the larvae was lost when the latex was washed or when theleaves were treated with a cysteine protease-inhibitor, indicating thatthe defense may be due to the high concentration of cysteine proteasesin the latex (Konno et al., 2004).

Proteases that target the cuticle are also important in insect control.The cuticle covers the entire outside of the insect as well as someinvaginations of internal structures. The cuticle is composed of a waxyepicuticle, an exocuticle and an endocuticle that consist of protein,lipid and chitin (Harrison and Bonning 2010). Fungal infection ofinsects by Metarhizium anisopliae and Beauveria bassiana occurs when thefungal spores germinate on the cuticle, forming structures forpenetration of the cuticle by a variety of enzymes, including proteases(Freimoser at al., 2003; Cho et al., 2006). One notable serine proteaseproduced by M. anisopliae, PR1A, digests the cuticle and plays anessential role in penetration (St. Leger et al. 1987). A clone of M.anisopliae was engineered to contain additional copies of the pr1a geneand showed 25% more kill of tobacco hornworm than the wild-type (St.Leger et al., 1996). B. basianna was also engineered to express the M.anisopliae PR1A protease and demonstrated increased toxicity of larvaeof the Masson's pine caterpillar, Dendrolimus punctatus, and the waxmoth, Galleria mellonella (Lu et al., 2008).

The basement membrane of insects consists of proteins that surround thetissue and contribute to a variety of functions from structural supportto barriers for viruses. Three potential basement membrane-degradingproteins were evaluated using Autographa californica multiplenucleopolyhedrovirus (AcMNPV). This baculovirus was engineered toexpress two vertebrate metalloproteases, rat stromelysin and humangeatinase A, as well as the fruit fly cathepsin L, ScathL. The ScathLprotease demonstrated the best baculovirus activity. The median survivaltime of infected tobacco budworm larvae was reduced by 50% when comparedto wild-type infected larvae (Harrison and Bonning, 2001). This datasupports the idea that proteases expressed in viruses have the abilityto access the basement membrane of insects, which generally functions asa barrier to viruses. A previous report identified two basement membraneproteins of imaginal discs of fruit fly larvae that are susceptible tohydrolysis by cathepsin L (Homma and Natori, 1996). Purified ScathLprotease was also toxic to a variety of insect pests when it wasinjected into the hemocoel. The purified protease demonstrated similarmelanization, mortality and hemolymph protease activity in lepidopteranlarvae as was seen ScathL expressed baculovirus infections (Li et al.,2008). Basement membrane damage is cause by purified ScathL proteaseboth in vivo and in vitro (Tang et al., 2007; Philip et al. 2007).

Arthropod predators have also been shown to contain basement membranecleaving proteases in their venom. One example is the parasitic wasp,Eulophus pennicornis, in which 3 metalloproteinases (EpMP1-3) wereidentified in the venom glands. Recombinant EpMP3 was injected into thehemocoel of Lacanobia oleracea larvae and resulted in significantmortality, or impaired development and growth in surviving larvae (Priceet al., 2009). Social aphid soldier nymphs produce a toxic cathepsin Bprotease (cysteine protease) in their intestines. The protease is orallyexcreted into enemies and demonstrates insecticidal activity (Kutsukakeet al., 2008).

A protease isolated from the bacterium, Xenorhabdus nematophilia, hasbeen shown to suppress antibacterial peptides involved in insect immuneresponse, making the insect susceptible to the pathogenetic process(Caldas et al., 2002). The enterobacterium, Photorhabdus luminscense,has been shown to be pathogenic to a broad spectrum of insects. Thegenome sequence of this bacterium identified genes related to toxicity,including proteases (Duchaud et al., 2003).

The use of proteases as insecticides has been of interest to plantmodifications as well. Basement-membrane degrading proteases have beencharacterized and engineered for transgenic insecticidal protocols, withthe goal of developing transgenic plants that are resistant to insectpests (U.S. Pat. No. 6,673,340, Harrison and Bonning, 2004). Proteasesin the gut of insects have been shown to affect the impact of Bacillusthuringiensis Cry insecticidal proteins. Some proteases activate Cryproteins by processing them from a protoxin to a toxic form. Insecttoxins have been modified to comprise proteolytic activation sites withthe goal of incorporating this modification into transformed plants,plant cells and seeds. Cleavage of these sites by the insect gutprotease results in an active insect toxin within the gut of the pest(U.S. Pat. No. 7,473,821, Abad et al., 2009).

Insecticidal Activity of Chitinases

Chitinases expedite insecticidal activity by puncturing the insectmidgut lining and degrading the insect cuticle. Degradation of thesemembranes exposes the insects to pathogens, to other insecticidalcompounds, and/or to plant defenses.

Chitinases hydrolyze the structural polysaccharide chitin, a linearhomopolymer of 2-acetamido-2-deoxy-D-glucopyranoside, linked byβ-1→4-linkages, which is a component of the exoskeleton and gut liningof insects. Chitinases are classified as either family 18 or family 19glycosyl hydrolases. Family 18 chitinases are widespread, found inbacteria, plants, and animals; while family 19 chitinases are mainlyfound in plants (Henrissat and Bairoch, 1993). In insects, Chitinasesplay a role in molting (Samuels and Reynolds, 1993, Merzendorfer andZimoch, 2003).

Chitinases alone show some insecticidal activity. Chitinase fromSerretia marcenscens was found to be toxic to seventh instar Galleriamellonella larvae (Lysenk, 1976). Transgenic plants which express insectchitinases have been shown to have increased resistance to insectpestss. Tobacco plants were transformed with cDNA encoding a Manducasexta chitinase. Leaves from these transgenic plants were infested withHeliothis virescens larvae. After 3 weeks it was found that chitinasepositive leaves had less larval biomass and feeding damage thanchitinase negative leaves. It is possible that the activity of thechitinases render insects more susceptible to plant defenses (Ding, etal., 1997).

Insect cuticles provide a physical barrier to protect the insect formpathogens or other environmental hazards, and are composed primarily ofchitin (Kramer, et al., 1995). Entomopathogenic fungi Metarhiziumanisopliae, Beauvaria bassiana, Beauvaria amorpha, Verticillium lecanii,and Aspergillus flavus all secrete chitinases to break down the cuticleand enter the insect host (St Leger, et al., 1986, 1992, Campos, et al.2005). According to Kim, et al., chitinase-containing supernatants ofBeauvaria bassina were toxic to Aphis gossypii adults. However, whenthese supernatants were treated with an excess of chitin to inhibit theactivity of the fungal chitinases, this mortality was significantlyreduced, suggesting that chitinase plays an integral role in breakingdown the cuticle and facilitating infection (Kim, et al. 2010).Chitinases have also been isolated from the venom of the endoparasiticwasp Chelonus sp., where they possibly help the venom penetrate thedefenses of chitin protected prey (Krishnan, et al., 1994).

The peritrophic membrane, which lines the insect midgut, is anotherprimarily-chitin-composed barrier that protects insects from pathogens.Any enzyme that can puncture this membrane has potential as abioinsecticide (Wang and Granados, 2001). Hubner, et al. demonstratedthat malarial parasites excrete chitinases to penetrate the peritrophicmembrane in mosquitoes (Hubner, et al., 1991), and Shahabuddin, et al.confirmed that inhibition of chitinase with allosamidin is sufficient toprevent the malarial parasite Plasmodium gallinaceum from crossing theperitrophic membrane of Anopheles freeborni. Also, the addition ofexogenous chitinase from Streptomyces griseus during the development ofthe Anopheles freeborni midgut prevented the formation of theperitrophic membrane (Shahabuddin, et al., 1993). This demonstrates thatchitinases can break down the peritrophic membrane. Regev, et al. usedE. coli to express Serratia marcescens endochitinase ChiA and confirmedwith electron microscopy that Spodoptera littoralis larvae exposed tothe endochitinase exhibited perforations in the peritrophic membrane(Regev, et al., 1996).

Because of the ability of chitinase to perforate the peritrophicmembrane, endochitinases have also been shown to increase theinsecticidal activity of Bacillus thuringiensis (Bt). Choristoneurafumiferana larvae reared on Agies balsamea treated with a mixture of adiluted commercial formulation of Bt and chitinase were killed morequickly than larvae reared on foliage treated with just Bt alone(Smirnoff, 1973). A mixture of a low concentration of Bt and S.marcenscens chitinase also resulted in higher mortality of Spodopteralittoralis larvae than Bt alone (Sheh et al., 1983). It is believed thatthis synergistic effect is due to puncturing of the peritrophic liningof the insect gut by the chitinase, facilitating the penetration of Btspores into the insect. (Smirnoff, 1973).

Yen-Tc, an ABC type protein that is both necessary and sufficient forthe entomopathogenicity of Yersinia entomophaga in the insect Costelytrazealandica, contains two family 18 chitinases, making it the firstinsecticidal toxin complex identified to incorporate chitinases. It ishypothesized that the chitinases are responsible for breaking downperitrophic membrane and exposing the midgut epithelial cells to thetoxin. However, the chitinases may only be active in regions of themidgut with a relatively neutral pH (Busby, 2012).

Chitinases are also integral to the activity of some insect viruses.Hatwin, et al. created mutants of the Autographa californicanucleopolyhedrovirus (AcMNPV) that lacked the gene for chitinase.Usually, this virus causes liquefaction of the host larvae, facilitatingthe spread of the virus. This liquefaction did not occur whenTrichoplusia ni larvae were infected with the chitinase negative virus.It was also confirmed that the AcMNPV chitinase is active under thealkaline conditions of the insect midgut (Hatwin, et al. 1997). Arecombinant version of the same Autographa californicanucleopolyhedrovirus that expressed a Haemaphysalis longicornischitinase was found to have bioarcaricidal activity againstHaemaphysalis longicornis nymphs (Assegna, et al. 2006).

Rhs-Like Genes Encode Insecticidal Toxins

The rhs (rearrangement hotspot) gene family was first identified in E.coli. These genes confer chromosomal rearrangements by homologousexchange (Lin et al., 1984). They are 2 to 12 kb in size and exhibit along core with a short tip. The core sequences are GC rich and highlyconserved, but the tip sequences are GC-poor and highly variable. Theyencode proteins that have a large core domain and a short C-terminal tipdomain. The protein core domain is hydrophilic and contains YD-repeats(Jackson et al., 2009). The Rhs proteins are capable of interacting withbacterial cell surfaces and binding to specific ligands (Wang et al.,1998). While the function of the Rhs proteins remains unknown (Hill etal., 1994), the structure is important because the YD repeats and highlyconserved sequences resemble rhs and rhs-like genes encodinginsecticidal toxins produced by bacteria.

Photorhabdus luminescens is a mutualistic symbiont of the nematodes fromthe Heterorhabditae family. The nematode infects the insect and injectsthe bacterium into the hemocoel of the insect. The bacterium thensecretes toxins that kill the insect (Frost et al., 1997). Bowen et al.(1998), purified a high molecular weight protein associated with oraland injectable insecticidal toxicity that targets insects. In anotherstudy, Bowen et al. (1998) used high performance liquid chromatographyto separate this protein into four toxin complexes (tc) termed, Tca,Tcb, Tcc, and Tcd encoded by the tc loci (Bowen et al., 1998).Waterfield et al. (2001) analyzed recombinant expression of the tc genesin E. coli to understand oral toxicity of Tc proteins. They found thatwithout tccC-like homologs, they could not recover oral toxicity in E.coli. These authors concluded that TccC is involved in activation oftoxin secretion. Furthermore, an amino acid sequence analysis revealedTccC and TccC-like proteins have a highly conserved core and highlyvariable extension. This structure bears resemblance to rhs-likeelements (Waterfield N R, Bowen D J, Fetherston J D, Perry R D, andffrench-Constant, RH, 2001). This similarity suggests that TccC-like andRhs proteins share an ancient role in toxin mobility and activation forthe Enterobacteriaceae family (ffrench-Constant, R et al, 2003).

Another microbe, Serratia entomophila, has insecticidal activity thattargets New Zealand grass grub, Costelytra zealandica, and causes amberdisease (Grimont et al., 1988). The virulence of S. entomophila islinked to a large plasmid called amber disease-associated plasmid(pADAP) (Glare et al., 1993). Hurst et al. analyzed the mutagenesis andthe nucleotide sequence of pADAP to understand how it conferspathogenicity to grass grub. They found that pADAP encodes three genesresponsible for the symptoms of amber disease, sepA, sepB, and sepC. Allthree genes are required for pathogenicity because a mutation in thesegenes abolishes amber disease. They illustrated that proteins encoded bythe sep genes are similar to the proteins encoded by the insecticidaltoxin complexes of P. luminescens. For example, the first 680 aminoacids of SepC and TccC show a strong similarity. Furthermore, thisregion resembles the rhs elements of E. coli. The sepC gene is smallerthan Rhs elements, but it encodes a hydrophilic protein core with nineRhs peptide variants. Based on the similarity between the sep and tcgenes, Hurst et al. concludes that these products are part of a newgroup of insecticidal toxins (Hurst et al., 2000).

Harada et al. discovered that, Pantoea stewartii ssp. DC283 is anaggressive pathogen that infects aphids (Harada et al., 1996). The aphidingests the bacterium and DC283 is able to aggregate in the gut andcause death of the aphid. Stavrinides et al. performed a mutagenesisscreen and discovered that the ucp1 (you cannot pass) locus isresponsible for the virulence of DC283. Analysis of the ucp1 genesequence revealed similarities to the Rhs protein family. ucp1 gene issmaller than the genes encoding RHS/YD proteins and does not have aligand binding YD repeat, but it has conserved 5′-cores, non-homologous3′ends, and it is a membrane bound protein. These structuralsimilarities suggest enteric plant colonizers have the genetic abilityto colonize insect hosts. Furthermore, the similarities between the ucp1and rhs genes suggest that rhs-like genes have potential insecticidalactivity (Stavrinides et al., 2010).

SUMMARY

The present disclosure provides the nucleotide sequence of the genome ofthe bacterium Chromobacterium subtsugae. Isolation and partialcharacterization of this bacterium is described, for example, in U.S.Pat. No. 7,244,607. Also provided are the nucleotide sequences of openreading frames in C. subtsugae; i.e., C. subtsugae gene sequences.Additionally provided are amino acid sequences of polypeptides encodedby the Chromobacterium subtsugae genome.

The present disclosure also provides isolated nucleic acids (e.g., DNA,RNA, nucleic acid analogues) comprising C. subtsugae genomic sequences,gene sequences, fragments thereof, and or mutant variants. Also providedare nucleic acid vectors (e.g., plasmid vectors, viral vectors),including expression vectors, comprising nucleic acids having C.subtsugae genome sequences, gene sequences, regulatory sequences and/orfragments thereof. Exemplary bacterial vectors include, but are notlimited to, Agrobacterium tumefaciens, Rhizobium sp. NGR234,Sinorhizobium meliloti, and Mesorhizobium loti.

Exemplary viral vectors include, but are not limited to, cauliflowermosaic virus (CaMV), pea early browning virus (PEBV), bean pod mottlevirus (BPMV), cucumber mosaic virus (CMV), apple latent spherical virus(ALSV), tobacco mosaic virus (TMV), potato virus X, brome mosaic virus(BMV) and barley stripe mosaic virus (BSMV).

Cells transfected with the foregoing nucleic acids or vectors are alsoprovided. Such cells can be plant cells, insect cells, mammalian cells,bacterial cells, or fungal cells (e.g., yeast). Plants comprising cells(plant or otherwise) that have been transfected with the foregoingnucleic acids or vectors, seeds from said plants, and the progeny ofsaid plants are also provided. Transfected bacterial cells can includeAgrobacteria (e.g., Agrobacterium tumefaciens), Rhizobium ,Sinorhizobium meliloti, and Mesorhizobium loti. Insect vectors (e.g.,Homalodisca vitripennis, the glassy-winged sharpshooter) comprisingnucleic acid vectors which themselves comprise C. subtsugae sequences,are also provided.

In additional embodiments, polypeptides encoded by the C. subtsugaegenome are provided. Functional fragments of C. subtsugae polypeptides,and conservatively substituted variants of C. subtsugae polypeptides,are also provided.

In further embodiments, plants comprising one or more isolated nucleicacids comprising C. subtsugae genomic sequences, gene sequences and/orfragments thereof are provided. These isolated nucleic acids can bepresent on the exterior of the plant or internally.

In additional embodiments, plants comprising one or more nucleic acidvectors, wherein said vector or vectors comprise C. subtsugae genomesequences, gene sequences and/or fragments thereof, are provided. Saidvectors can be present on the exterior of the plant or internally.

In yet additional embodiments, plants comprising one or more C.subtsugae polypeptides are provided. Said C. subtsugae polypeptides canbe present on the exterior of the plant or internally.

Also provided are plants comprising one or more functional fragmentsand/or one or more conservatively substituted variants of a C. subtsugaepolypeptide or polypeptides. Said fragments and/or conservativelysubstituted variants can be present on the exterior of the plant orinternally.

Progeny of the aforementioned plants are also provided. In addition,seeds from the aforementioned plants, and from their progeny, areprovided.

Also disclosed herein are methods for controlling pests; e.g., methodsfor modulating pest infestation in a plant. Such pests can be, forexample, insects, fungi, nematodes, mites, moths or aphids. The methodsinclude application of a nucleic acid comprising a C. subtsugae genomesequence, gene sequence, or fragment thereof to a plant, eitherinternally or externally. Additional methods include application of a C.subtsugae polypeptide, or fragment thereof, or conservativelysubstituted variant thereof, to a plant, either internally orexternally.

Also provided are pesticidal (e.g., insecticidal) compositionscomprising nucleic acids and/or polypeptides encoded by the C. subtsugaegenome. Such compositions can optionally include other insecticides orpesticides, either naturally-occurring or man-made.

Also provided is a computer-readable medium comprising the sequenceinformation of any of the nucleotide or amino acid sequences disclosedherein (i.e., any of SEQ ID NOs 1-8960) or any fragment thereof. Alsoprovided are computerized systems and computer program productscontaining the nucleic acids and polypeptide sequences disclosed hereinon a computer-readable medium, for use in, for example, sequenceanalysis and comparison.

Accordingly, disclosed herein, inter alia, are the followingembodiments:

-   -   1. An isolated nucleic acid having the sequence of any one of        SEQ ID NOs: 1-4533. Nucleic acids as disclosed herein can be        DNA, RNA, or any nucleic acid analogue known in the art.    -   2. An isolated nucleic acid having 10 or more contiguous        nucleotides of the sequence of SEQ ID NO: 1. Nucleic acids as        disclosed herein can be DNA, RNA, or any nucleic acid analogue        known in the art.    -   3. An isolated nucleic acid having 10 or more contiguous        nucleotides of the sequence of any one of SEQ ID NOs: 2-4533.        Nucleic acids as disclosed herein can be DNA, RNA, or any        nucleic acid analogue known in the art.    -   4. An isolated nucleic acid comprising a C. subtsugae regulatory        sequence.    -   5. The nucleic acid of embodiment 4, wherein the regulatory        sequence is a promoter or an operator.    -   6. The nucleic acid of embodiment 4, wherein the regulatory        sequence is a transcription terminator.    -   7. An isolated nucleic acid comprising a sequence that is        complementary to the sequence of any of the nucleic acids of        embodiments 1-6.    -   8. A nucleic acid vector comprising the isolated nucleic acid of        any of embodiments 1-7.    -   9. The nucleic acid vector of embodiment 8, wherein the vector        is an expression vector.    -   10. An isolated polypeptide having the sequence of any one of        SEQ ID NOs: 4534-8960.    -   11. An isolated polypeptide having 10 or more contiguous amino        acids of the sequence of any one of SEQ ID NOs: 4534-8960.    -   12. A functional fragment of the polypeptide of embodiment 10.    -   13. A conservatively substituted variant of the polypeptide of        embodiment 10.    -   14. A polypeptide comprising an amino acid sequence having at        least 75% homology to the sequences of any of embodiments 10-13.    -   15. An isolated nucleic acid encoding a polypeptide according to        any of embodiments 10-14.    -   16. An isolated nucleic acid comprising a sequence that is        complementary to the sequence of the nucleic acid of embodiment        15.    -   17. An isolated nucleic acid comprising a sequence having at        least 75% homology to the sequences of any of embodiments 1-7,        15 or 16, or to either of the vectors of embodiments 8 or 9.    -   18. A cell comprising the isolated nucleic acid of any of        embodiments 1-7, 15 or 16, or with the nucleic acid vector of        either of embodiments 8 or 9. Such cells can be, e.g., plant        cells, insect cells, bacterial cells (e.g., Agrobacterium) or        fungal cells (e.g., yeast).    -   19. A plant comprising one or more cells according to embodiment        18.    -   20. The plant of embodiment 19 wherein the cell is a plant cell.    -   21. The plant of embodiment 20 wherein the cell is of the same        species as the plant.    -   22. The progeny of the plant of any of embodiments 19-21.    -   23. A seed from the plant of any of embodiments 19-22.    -   24. A plant comprising one or more nucleic acids according to        any of embodiments 1-7 or 15-17, or one or more of the nucleic        acid vectors of embodiments 8 or 9.    -   25. The plant of embodiment 24, wherein the nucleic acid or        vector is present on the exterior of the plant.    -   26. The plant of embodiment 24, wherein the nucleic acid or        vector is present in the interior of the plant.    -   27. The plant of embodiment 26, wherein the nucleic acid or        vector is intracellular.    -   28. The progeny of the plant of embodiment 27.    -   29. A seed from the plant of either of embodiments 27 or 28.    -   30. A plant comprising one or more polypeptides according to any        of embodiments 10-14.    -   31. The plant of embodiment 30, wherein the polypeptide is        present on the exterior of the plant.    -   32. The plant of embodiment 30, wherein the polypeptide is        present in the interior of the plant.    -   33. The plant of embodiment 32, wherein the polypeptide is        intracellular.    -   34. A method for modulating pest infestation in a plant, the        method comprising contacting a plant or a plant part with a        composition comprising one or more nucleic acids according to        any of embodiments 1-7 or 15-17, or one or more of the nucleic        acid vectors of embodiments 8 or 9, or one or more polypeptides        according to any of embodiments 10-14.    -   35. The method of embodiment 34, wherein said contacting        comprises one of the following:    -   (a) applying the composition to the plant;    -   (b) applying the composition to the substrate in which the plant        is growing;    -   (c) applying the composition to the root zone of the plant; or    -   (d) dipping the roots of the plant into the composition prior to        planting.    -   36. The method of embodiment 35, wherein said applying comprises        one of the following:    -   (a) applying the composition to plants or turf as a soil or root        drench;    -   (b) applying via irrigation; or    -   (c) contacting a seed with the composition.    -   37. The method of embodiment 34, wherein the pest is selected        from the group consisting of insects, fungi, nematodes, bacteria        and mites.    -   38. The method of embodiment 34, wherein the composition is        applied to the exterior of the plant.    -   39. The method of embodiment 34, wherein the composition is        applied to the interior of the plant.    -   40. The method of embodiment 39, wherein the nucleic acid or the        vector or the polypeptide is intracellular.    -   41. A pesticidal composition comprising one or more nucleic        acids according to any of embodiments 1-7 or 15-17, or a vector        according to either of embodiments 8 or 9.    -   42. A pesticidal composition comprising one or more polypeptides        according to any of embodiments 10-14.    -   43. The pesticidal composition of either of embodiments 41 or        42, wherein the composition is an insecticide.    -   44. The pesticidal composition of any of embodiments 41-43,        further comprising a second pesticide.    -   45. The pesticidal composition of embodiment 44, wherein the        second pesticide is an insecticide.    -   46. A computer-readable medium comprising the sequence        information of any of SEQ ID NOs: 1-8960.    -   47. A computer-readable medium comprising the sequence        information of any of the nucleic acids of embodiments 1-7 or        15-17, or the vectors of either of embodiments 8 or 9.    -   48. A computer-readable medium comprising the sequence        information of any of the polypeptides of embodiments 10-14.    -   49. A nucleic acid that hybridizes, under high-stringency        conditions, to the nucleic acid of any of embodiments 1-7 or        15-17.    -   50. The nucleic acid of any of embodiments 1-7 or 15-17, further        comprising a heterologous nucleotide sequence.    -   51. The nucleic acid of embodiment 50, wherein said heterologous        nucleotide sequence is a regulatory sequence.    -   52. The nucleic acid of embodiment 50, wherein said heterologous        nucleotide sequence encodes a heterologous polypeptide.    -   53. The polypeptide of any of embodiments 10-14, further        comprising a heterologous amino acid sequence.    -   54. An antibody that binds to the polypeptide of any of        embodiments 10-14.

DETAILED DESCRIPTION

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields ofagriculture, plant molecular biology, entomology, cell biology,molecular biology, biochemistry, recombinant DNA and related fields asare within the skill of the art. Such techniques are described in theliterature and thereby available to those of skill in the art. See, forexample, Alberts, B. et al., “Molecular Biology of the Cell,” 5^(th)edition, Garland Science, New York, N.Y., 2008; Voet, D. et al.“Fundamentals of Biochemistry: Life at the Molecular Level,” 3^(rd)edition, John Wiley & Sons, Hoboken, N.J., 2008; Sambrook, J. et al.,“Molecular Cloning: A Laboratory Manual,” 3^(rd) edition, Cold SpringHarbor Laboratory Press, 2001; Ausubel, F. et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, New York, 1987 and periodicupdates; Glover, DNA Cloning: A Practical Approach, volumes I and II,IRL Press (1985), volume III, IRL Press (1987); Perbal, A PracticalGuide to Molecular Cloning, John Wiley & Sons (1984); Rigby (ed.), Theseries “Genetic Engineering” (Academic Press); Setlow & Hollaender(eds.), The series “Genetic Engineering: Principles and Methods,” PlenumPress; Gait (ed.), Oligonucleotide Synthesis: A Practical Approach, IRLPress (1984, 1985); Eckstein (ed.) Oligonucleotides and Analogues: APractical Approach, IRL Press (1991); Hames & Higgins, Nucleic AcidHybridization: A Practical Approach, IRL Press (1985); Hames & Higgins,Transcription and Translation: A Practical Approach, IRL Press (1984);B. Buchanan, W. Gruissem & R. Jones (eds.) “Biochemistry and MolecularBiology of Plants,” Wiley (2002) and the series “Methods in Enzymology,”Academic Press, San Diego, Calif. The disclosures of all of theforegoing references are incorporated by reference in their entiretiesfor the purpose of describing methods and compositions in the relevantarts.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is included therein. Smaller ranges are also included. Theupper and lower limits of these smaller ranges are also includedtherein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” and “the” include plural references unless thecontext clearly dictates otherwise.

Polynucleotides and Oligonucleotides

A polynucleotide is a polymer of nucleotides, and the term is meant toembrace smaller polynucleotides (fragments) generated by fragmentationof larger polynucleotides. The terms polynucleotide and nucleic acidencompass both RNA and DNA, as well as single-stranded anddouble-stranded polynucleotides and nucleic acids. Polynucleotides alsoinclude modified polynucleotides and nucleic acids, containing suchmodifications of the base, sugar or phosphate groups as are known in theart.

An oligonucleotide is a short nucleic acid, generally DNA and generallysingle-stranded. Generally, an oligonucleotide will be shorter than 200nucleotides, more particularly, shorter than 100 nucleotides, mostparticularly, 50 nucleotides or shorter.

Modified bases and base analogues, e.g., those able to form Hoogsteenand reverse Hoogsteen base pairs with the naturally-occurring bases, areknown in the art. Examples include, but are not limited to,8-oxo-adenosine, pseudoisocytidine, 5-methyl cytidine, inosine,2-aminopurine and various pyrrolo- and pyrazolopyrimidine derivatives.Similarly, modified sugar residues or analogues, for example2′-O-methylribose or peptide nucleic acid backbones, can also form acomponent of a modified base or base analogue. See, for example, Sun andHelene (1993) Curr. Opin. Struct. Biol. 3:345-356. Non-nucleotidemacromolecules capable of any type of sequence-specific interaction witha polynucleotide are useful in the methods and compositions disclosedherein. Examples include, but are not limited to, peptide nucleic acids,minor groove-binding agents and antibiotics. New modified bases, baseanalogues, modified sugars, sugar analogues, modified phosphates andphosphate analogues capable of participating in duplex or triplexformation are available in the art, and are useful in the methods andcompositions disclosed herein.

Homology and Identity of Nucleic Acids and Polypeptides

“Homology” or “identity” or “similarity” as used herein in the contextof nucleic acids and polypeptides refers to the relationship between twopolypeptides or two nucleic acid molecules based on an alignment of theamino acid sequences or nucleic acid sequences, respectively. Homologyand identity can each be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. For example, a“reference sequence” can be compared with a “test sequence.” When aposition in the reference sequence is occupied by the same base or aminoacid at an equivalent position in the test sequence, then the moleculesare identical at that position; when the equivalent position is occupiedby a similar amino acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. The relatedness of two sequences, whenexpressed as a percentage of homology/similarity or identity, is afunction of the number of identical or similar amino acids at positionsshared by the sequences being compared. In comparing two sequences, theabsence of residues (amino acids or nucleic acids) or presence of extraresidues, in one sequence as compared to the other, also decreases theidentity and homology/similarity.

As used herein, the term “identity” refers to the percentage ofidentical nucleotide or amino acid residues at corresponding positionsin two or more sequences when the sequences are aligned to maximizesequence matching, i.e., taking into account gaps and insertions.Identity can be readily calculated by known methods, including but notlimited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988). Methods to determine identity aredesigned to give the highest degree of match between the sequencestested. Moreover, methods to determine identity are codified in publiclyavailable computer programs. Computer program methods to determineidentity between two sequences include, but are not limited to, the GCGprogram package (Devereux et al. (1984) Nucleic Acids Research 12:387),BLASTP, BLASTN, and FASTA (Altschul et al. (1990) J. Molec. Biol.215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).The BLAST X program is publicly available from NCBI and other sources.See, e.g., BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda,Md. 20894; Altschul et al. (1990) J. Mol. Biol. 215:403-410. The wellknown Smith-Waterman algorithm can also be used to determine identity.

For sequence comparison, typically one sequence acts as a referencesequence, to which one or more test sequences are compared. Sequencesare generally aligned for maximum correspondence over a designatedregion, e.g., a region at least about 20, 25, 30, 35, 40, 45, 50, 55,60, 65 or more amino acids or nucleotides in length, and the region canbe as long as the full-length of the reference amino acid sequence orreference nucleotide sequence. When using a sequence comparisonalgorithm, test and reference sequences are input into a computerprogram, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Examples of algorithms that are suitable for determining percentsequence identity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (1990) J. Mol. Biol. 215:403-410 andAltschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information at www.ncbi.nlm.nih.gov(visited Dec. 27, 2012). Further exemplary algorithms include ClustalW(Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680), available atwww.ebi.ac.uk/Tools/clustalw/index.html (visited Dec. 27, 2012).

Sequence identity between two nucleic acids can also be described interms of annealing, reassociation, or hybridization of twopolynucleotides to each other, mediated by base-pairing. Hybridizationbetween polynucleotides proceeds according to well-known andart-recognized base-pairing properties, such that adenine base-pairswith thymine or uracil, and guanine base-pairs with cytosine. Theproperty of a nucleotide that allows it to base-pair with a secondnucleotide is called complementarity. Thus, adenine is complementary toboth thymine and uracil, and vice versa; similarly, guanine iscomplementary to cytosine and vice versa. An oligonucleotide orpolynucleotide which is complementary along its entire length with atarget sequence is said to be perfectly complementary, perfectlymatched, or fully complementary to the target sequence, and vice versa.Two polynucleotides can have related sequences, wherein the majority ofbases in the two sequences are complementary, but one or more bases arenoncomplementary, or mismatched. In such a case, the sequences can besaid to be substantially complementary to one another. If twopolynucleotide sequences are such that they are complementary at allnucleotide positions except one, the sequences have a single nucleotidemismatch with respect to each other.

Conditions for hybridization are well-known to those of skill in the artand can be varied within relatively wide limits. Hybridizationstringency refers to the degree to which hybridization conditionsdisfavor the formation of hybrids containing mismatched nucleotides,thereby promoting the formation of perfectly matched hybrids or hybridscontaining fewer mismatches; with higher stringency correlated with alower tolerance for mismatched hybrids. Factors that affect thestringency of hybridization include, but are not limited to,temperature, pH, ionic strength, and concentration of organic solventssuch as formamide and dimethylsulfoxide. As is well known to those ofskill in the art, hybridization stringency is increased by highertemperatures, lower ionic strengths, and lower solvent concentrations.See, for example, Ausubel et al., supra; Sambrook et al., supra; M. A.Innis et al. (eds.) PCR Protocols, Academic Press, San Diego, 1990; B.D. Hames et al. (eds.) Nucleic Acid Hybridisation: A Practical Approach,IRL Press, Oxford, 1985; and van Ness et al., (1991) Nucleic Acids Res.19:5143-5151.

Thus, in the formation of hybrids (duplexes) between twopolynucleotides, the polynucleotides are incubated together in solutionunder conditions of temperature, ionic strength, pH, etc., that arefavorable to hybridization, i.e., under hybridization conditions.Hybridization conditions are chosen, in some circumstances, to favorhybridization between two nucleic acids having perfectly-matchedsequences, as compared to a pair of nucleic acids having one or moremismatches in the hybridizing sequence. In other circumstances,hybridization conditions are chosen to allow hybridization betweenmismatched sequences, favoring hybridization between nucleic acidshaving fewer mismatches.

The degree of hybridization between two polynucleotides, also known ashybridization strength, is determined by methods that are well-known inthe art. A preferred method is to determine the melting temperature(T_(m)) of the hybrid duplex. This is accomplished, for example, bysubjecting a duplex in solution to gradually increasing temperature andmonitoring the denaturation of the duplex, for example, by absorbance ofultraviolet light, which increases with the unstacking of base pairsthat accompanies denaturation. T_(m) is generally defined as thetemperature midpoint of the transition in ultraviolet absorbance thataccompanies denaturation. Alternatively, if T_(m)s are known, ahybridization temperature (at fixed ionic strength, pH and solventconcentration) can be chosen that is below the T_(m) of the desiredduplex and above the T_(m) of an undesired duplex. In this case,determination of the degree of hybridization is accomplished simply bytesting for the presence of duplex polynucleotide.

Hybridization conditions are selected following standard methods in theart. See, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y. For example,hybridization reactions can be conducted under stringent conditions. Anexample of stringent hybridization conditions is hybridization at 50° C.or higher in 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Another example of stringent hybridization conditions is overnightincubation at 42° C. in a solution: 50% formamide, 5×SSC (0.75 M NaCl,75 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), followed bywashing in 0.1×SSC at about 65° C. Optionally, one or more of 5×Denhardt's solution, 10% dextran sulfate, and/or 20 mg/ml heterologousnucleic acid (e.g., yeast tRNA, denatured, sheared salmon sperm DNA) canbe included in a hybridization reaction. Stringent hybridizationconditions are hybridization conditions that are at least as stringentas the above representative conditions, where conditions are consideredto be at least as stringent if they are at least about 80% as stringent,typically at least 90% as stringent as the above specific stringentconditions.

The term “substantially identical” refers to identity between a firstamino acid sequence that contains a sufficient or minimum number ofamino acid residues that are i) identical to, or ii) conservativesubstitutions of, aligned amino acid residues in a second amino acidsequence such that the first and second amino acid sequences share acommon structural domain and/or common functional activity. For example,amino acid sequences that contain a common structural domain having atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%identity to an amino acid sequence as disclosed herein (i.e., SEQ IDNOs: 4534-8960) are termed substantially identical. In the context ofnucleotide sequence, the term “substantially identical” is used hereinto refer to a first nucleic acid sequence that contains a sufficient orminimum number of nucleotides that are identical to aligned nucleotidesin a second nucleic acid sequence such that the first and secondnucleotide sequences encode a polypeptide having common functional orstructural activity, or encode a common structural polypeptide domain ora common functional polypeptide activity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. A reference nucleotide or amino acidsequence (e.g., a sequence as disclosed herein) is used as a “querysequence” to perform a search against public databases to, for example,identify other family members, related sequences or homologues. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a referencenucleotide sequence. BLAST amino acid searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a reference amino acid sequence. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing the BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and BLAST) can beused (see the world wide web at: ncbi.nlm.nih.gov).

Nucleic acids and polynucleotides of the present disclosure encompassthose having an nucleotide sequence that is at least 75%, at least 80%,at least 90%, at least 95%, at least 99% or 100% identical to any of SEQID NOs: 2-4533.

Nucleotide analogues and amino acid analogues are known in the art.Accordingly, nucleic acids (i.e., SEQ ID NOs: 1-4533X) comprisingnucleotide analogues and polypeptides (i.e., SEQ ID NOs: 4534-8960)comprising amino acid analogues are also encompassed by the presentdisclosure.

Conservative Substitutions and Functional Fragments

In comparing amino acid sequences, residue positions which are notidentical can differ by conservative amino acid substitutions.Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. With respect to a referencepolypeptide sequence, a test polypeptide sequence that differs only byconservative substitutions is denoted a “conservatively substitutedvariant” of the reference sequence.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. See Ausubel et al., supra. The ability of aprotein to interact with another protein can be determined, for example,by co-immunoprecipitation, two-hybrid assays or complementation, eithergenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245 246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

Typically, a functional fragment retains at least 50% of the activity orfunction of the polypeptide. In some embodiments, a functional fragmentretains at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99% or 100% of the activity or function of thepolypeptide.

A functional fragment of a polypeptide can include conservative aminoacid substitutions (with respect to the native polypeptide sequence)that do not substantially alter the activity or function of thepolypeptide. The term “conservative amino acid substitution” refers togrouping of amino acids on the basis of certain common structures and/orproperties. With respect to common structures, amino acids can begrouped into those with non-polar side chains (glycine, alanine, valine,leucine, isoleucine, methionine, proline, phenylalanine and tryptophan),those with uncharged polar side chains (serine, threonine, asparagine,glutamine, tyrosine and cysteine) and those with charged polar sidechains (lysine, arginine, aspartic acid, glutamic acid and histidine). Agroup of amino acids containing aromatic side chains includesphenylalanine, tryptophan and tyrosine. Heterocyclic side chains arepresent in proline, tryptophan and histidine. Within the group of aminoacids containing non-polar side chains, those with short hydrocarbonside chains (glycine, alanine, valine. leucine, isoleucine) can bedistinguished from those with longer, non-hydrocarbon side chains(methionine, proline, phenylalanine, tryptophan). Within the group ofamino acids with charged polar side chains, the acidic amino acids(aspartic acid, glutamic acid) can be distinguished from those withbasic side chains (lysine, arginine and histidine).

A functional method for defining common properties of individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz, G. E.and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag,1979). According to such analyses, groups of amino acids can be definedin which amino acids within a group are preferentially substituted forone another in homologous proteins, and therefore have similar impact onoverall protein structure (Schulz, G. E. and R. H. Schirmer, supra).According to this type of analysis, conservative amino acidsubstitution” refers to a substitution of one amino acid residue foranother sharing chemical and physical properties of the amino acid sidechain (e.g., charge, size, hydrophobicity/hydrophilicity). Following areexamples of amino acid residues sharing certain chemical and/or physicalproperties:

-   -   (i) amino acids containing a charged group, consisting of Glu,        Asp, Lys, Arg and His,    -   (ii) amino acids containing a positively-charged group,        consisting of Lys, Arg and His,    -   (iii) amino acids containing a negatively-charged group,        consisting of Glu and Asp,    -   (iv) amino acids containing an aromatic group, consisting of        Phe, Tyr and Trp,    -   (v) amino acids containing a nitrogen ring group, consisting of        His and Trp,    -   (vi) amino acids containing a large aliphatic non-polar group,        consisting of Val, Leu and Ile,    -   (vii) amino acids containing a slightly-polar group, consisting        of Met and Cys,    -   (viii) amino acids containing a small-residue group, consisting        of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro,    -   (ix) amino acids containing an aliphatic group consisting of        Val, Leu, Ile, Met and Cys, and    -   (x) amino acids containing a hydroxyl group consisting of Ser        and Thr.

Certain “conservative substitutions” may include substitution within thefollowing groups of amino acid residues: gly, ala; val, ile, leu; asp,glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Thus, as exemplified above, conservative substitutions of amino acidsare known to those of skill in this art and can be made generallywithout altering the biological activity or function of the resultingmolecule. Those of skill in this art also recognize that, in general,single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity. See, e.g.,Watson, et al., “Molecular Biology of the Gene,” 4th Edition, 1987, TheBenjamin/Cummings Pub. Co., Menlo Park, Calif., p. 224.

Polypeptides of the present disclosure encompass those having 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 or more amino acid substitutions compared to anamino acid sequence as set forth in SEQ ID NOs: 4534-8960, e.g.,conservative amino acid substitutions. Amino acid residues that can besubstituted can be located at residue positions that are not highlyconserved. The ordinarily skilled artisan will appreciate that, based onlocation of the active sites and/or on homology to related proteins, aprotein will tolerate substitutions, deletions, and/or insertions atcertain of its amino acid residues, without significant change in itsoverall physical and chemical properties.

Polypeptides of the present disclosure encompass those having an aminoacid sequence that is at least 75%, at least 80%, at least 90%, at least95%, at least 99% or 100% identical to any of the polypeptides shown inSEQ ID NOs: 4534-8960.

C. subtsugae Nucleic Acids

The present disclosure provides the entire nucleotide sequence of the C.subtsugae genome (SEQ ID NO:1). This genome contains 4,705,004 bp, whichincludes 4,415 protein-coding sequences (i.e., open reading frames orORFs) and 118 functional RNA sequences.

Also provided are nucleotide sequences of open reading frames (ORFs)encoding C. subtsugae genes and nucleotide sequences of functional RNAmolecules (e.g., rRNAs, tRNAs) (SEQ ID NOs: 2-4533) as disclosed inTable 1. Nucleic acids comprising these sequences are also provided.Fragments of the C. subtsugae genome and/or fragments of C. subtsugaegene sequences are also provided. Such fragments are 10 or more, 25 ormore, 50 or more, 75 or more, 100 or more 200 or more, 500 or more, or1,000 or more nucleotides in length. Nucleic acids having a sequencethat is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 99.9% identical to the aforementioned sequences are alsoprovided. The nucleic acids disclosed herein can be either DNA or RNA,and can be either single-stranded or double-stranded. Nucleic acidscomprising nucleotide sequences that are complementary to theaforementioned sequences are also provided, as are nucleic acids thathybridize to the aforementioned nucleic acids under stringentconditions.

Fragments of the C. subtsugae genome that encode polypeptides (i.e.,open reading frames or ORFs) are provided. C. subtsugae ORFs encodesecreted proteins that include, inter alia, proteases, chitinases, rhs(rearrangement hotspot) proteins, lipases, phospholipases, esterases,toxins, proteins involved in iron metabolism, proteins involved inphosphate metabolism, proteins involved in plant growth, and proteinsinvolved in biosynthesis of fimbria and pili. Genome fragments thatencode protein clusters, e.g., those involved in non-ribosomal peptidesynthesis (NRPS), and other biosynthetic clusters, are also provided. C.subtsugae ORFs also encode transmembrane proteins that include, interalia, transporters, proteases, toxins, antibiotics and proteins thatconfer antibiotic resistance. Additional fragments of the C. subtsugaegenome encode functional RNA molecules, such as, for example, rRNAs andtRNAs. Yet additional fragments of the C. subtsugae genome comprisetranscriptional and translational regulatory sequences such aspromoters, operators, terminators ribosome binding sites, etc.

Additional C. subtsugae ORFs encode proteins that confer insecticideactivity, miticide activity, nematicide activity, algaecide activity orcan be used in bioremediation methods.

Additional C. subtsugae ORFs encode proteins that participate in thesynthesis of metabolites that confer insecticide activity, miticideactivity, nematicide activity, algaecide activity or can be used inbioremediation methods.

The subject nucleic acids can optionally comprise heterologousnucleotide sequences. Such heterologous nucleotide sequences can beregulatory sequences, such as promoters, operators, enhancers,terminators and the like; or can encode heterologous amino acid (i.e.,polypeptide) sequences.

For example, a heterologous regulatory sequence can be joined inoperative linkage to a C. subtsugae protein-encoding sequence (i.e. ORF)to provide regulated expression of a C. subtsugae protein. Suchconstructs can be used, e.g., for regulated expression and/oroverexpression of pesticidal C. subtsugae proteins (e.g., chitinases,lipases, proteases) in a host cell. Such constructs can also be used forregulated expression and/or overexpression of an enzyme encoded by theC. subtsugae genome that catalyzes the synthesis of a pesticidalmetabolite (or an intermediate in the synthesis of a pesticidalmetabolite). Host cells can be chosen to facilitate expression and/orpurification of cloned C. subtsugae proteins.

In additional embodiments, a C. subtsugae regulatory sequence can bejoined in operative linkage with a heterologous coding sequence (e.g.,ORF) to provide regulated expression of a heterologous protein in, e.g.,C. subtsugae or another host. Such a protein can be for example, apesticidal protein not encoded by the C. subtsugae genome or an enzymethat catalyzes the synthesis of a pesticidal metabolite. Such an enzymecan be encoded by the C. subtsugae genome or encoded by a heterologousorganism.

The present disclosure also provides polynucleotides comprising anucleotide sequence encoding any of the polypeptide sequences disclosedherein. Such a polynucleotide has a nucleotide sequence that is at least70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% or 100%) identical to a contiguoussequence of a nucleic acid that encodes any of the polypeptidesdisclosed herein. The percentage identity is based on the shorter of thesequences compared. Well known programs such as BLASTN (2.0.8) (Altschulet al. (1997) Nucl. Acids. Res. 25:3389-3402) using default parametersand no filter can be employed to make a sequence comparison. Nucleicacid sequence identity (e.g. between two different polynucleotidesencoding identical amino acid sequences) can be lower than the percentof amino acid sequence identity due to degeneracy of the genetic code.

Examples of nucleic acid sequences in a polynucleotide encoding apolypeptide of the present disclosure can be found among SEQ ID NOs:2-4533. These nucleic acid sequences can also be provided in anexpression vector (see below).

C. subtsugae Polypeptides and Proteins

The present disclosure provides the amino acid sequences of proteinsencoded by the C. subtsugae genome, as well as polypeptides comprisingsaid amino acid sequences (i.e., SEQ ID NOs: 4534-8960). Functionalfragments and conservatively-substituted variants of said polypeptidesare also provided. In addition, fragments of the polypeptides disclosedherein that do not retain function are also provided and are useful,e.g., as epitopes for production of antibodies. Such fragments are 4 ormore, 10 or more, 25 or more, 50 or more, 75 or more, 100 or more 200 ormore, 500 or more, or 1,000 or more amino acids in length.

The present disclosure also provides a polypeptide comprising an aminoacid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to a contiguous sequenceof a polypeptide as disclosed herein. The percentage identity is basedon the shorter of the sequences compared. Methods for determining degreeof polypeptide sequence identity are well-known in the art.

The subject polypeptides can include amino acid sequences derived fromany of SEQ ID NOs: 4534-8960 further comprising heterologous amino acidsequences. Such polypeptides can be fusion proteins, such as a fusionprotein containing epitope tags, purification tags, and/or detectablelabels. A fusion protein can optionally include a linker sequencebetween the heterologous sequences and the C. subtsugae amino acidsequence. Methods for producing fusion proteins are well-known in theart. Other heterologous elements and exemplary fusion proteins aredescribed in more detail below.

Exemplary polypeptides containing heterologous elements may include mycand/or His₆ tags and may optionally include flanking linker sequences.

Polypeptides of the present disclosure further encompass those that arejoined to a reporter polypeptide, e.g., a fluorescent protein, and/orconjugated to a molecule. The molecule conjugated to the polypeptide canbe a carrier molecule or a moiety that facilitates delivery and/orincreases the half-life of the subject polypeptide.

Polypeptides of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis). The subject polypeptide can be prepared bysolid-phase synthesis methods well-known in the art, (e.g., Fmoc- ort-Boc chemistry), such as those described by Merrifield (1963) J. Am.Chem. Soc. 85:2149 and Methods in Molecular Biology, Vol 35: PeptideSynthesis Protocols.

It should be noted that the polypeptides of the present disclosure canalso contain additional elements, such as a detectable label, e.g., aradioactive label, a fluorescent label, a biotin label, animmunologically detectable label (e.g., a hemagglutinin (HA) tag, apoly-Histidine tag) and the like. Additional elements can be provided(e.g., in the form of fusion polypeptides) to facilitate expression(e.g. N-terminal methionine and/or a heterologous signal sequence tofacilitate expression in host cells), and/or isolation (e.g., biotintag, immunologically detectable tag) of the polypeptides of thedisclosure through various methods. The polypeptides can also optionallybe immobilized on a support through covalent or non-covalent attachment.

Isolation and purification of the subject polypeptides can beaccomplished according to methods known in the art. The term “isolated”is intended to mean that a compound (e.g. polypeptide or polynucleotide)is separated from all or some of the components that accompany it innature. “Isolated” also refers to the state of a compound separated fromall or some of the components that accompany it during manufacture(e.g., chemical synthesis, recombinant expression, culture medium, andthe like).

For example, a polypeptide according to the present disclosure can beisolated from a lysate of cells that have been genetically modified toexpress the subject polypeptide, from a cell culture medium, or from asynthetic reaction mixture. Isolation can additionally be achieved byimmunoaffinity purification, which generally involves contacting asample with an antibody (optionally immobilized) that specifically bindsto an epitope of the polypeptide, washing to remove non-specificallybound material, and eluting specifically bound polypeptide. Isolatedpolypeptide can be further purified by dialysis and other methodsnormally employed in protein purification, e.g. metal chelatechromatography, ion-exchange, and size exclusion.

Secreted Proteins

C. subtsugae sequences were examined for the presence of a signalsequence, indicative of secreted proteins. C. subtsugae proteinscontaining a signal sequence are disclosed in this section.

Tables 2-4 provide examples of C. subtsugae ORFs encoding potentiallysecreted proteins known to act against insects.

TABLE 2 Proteases CDS ID Function fig|6666666.22288.peg.160 Zn-dependentprotease with chaperone function fig|6666666.22288.peg.173 Probableendonuclease fig|6666666.22288.peg.176 Bacterial leucyl aminopeptidase(EC 3.4.11.10) fig|6666666.22288.peg.1274 Putative peptidasefig|6666666.22288.peg.1991 Probable protease fig|6666666.22288.peg.1992Probable protease fig|6666666.22288.peg.2084 HtrA protease/chaperoneprotein fig|6666666.22288.peg.2155 Putative extracellular serineprotease fig|6666666.22288.peg.2281 Cell wall endopeptidase, familyM23/M37 fig|6666666.22288.peg.2516 Probable Peptidasefig|6666666.22288.peg.2583 LasA protease precursorfig|6666666.22288.peg.2594 Dipeptidylaminopeptidases/acylaminoacyl-peptidases fig|6666666.22288.peg.3226Tricorn protease homolog (EC 3.4.21.-) fig|6666666.22288.peg.3193Murein-DD-endopeptidase (EC 3.4.99.-) fig|6666666.22288.peg.3559 Prolylendopeptidase (EC 3.4.21.26) fig|6666666.22288.peg.3563 Probableprotease precursor fig|6666666.22288.peg.3576 Possible periplasmicaspartyl protease fig|6666666.22288.peg.3897 Putative protease ydgD (EC3.4.21.-) fig|6666666.22288.peg.4266 Zinc protease( EC:3.4.99.- )fig|6666666.22288.peg.4323 Probable metallopeptidasefig|6666666.22288.peg.175 Vibriolysin, extracellular zinc protease (EC3.4.24.25) fig|6666666.22288.peg.452 Exported zinc metalloprotease YfgCprecursor fig|6666666.22288.peg.1216 D-alanyl-D-alanine carboxypeptidase(EC 3.4.16.4) fig|6666666.22288.peg.2125 Metallopeptidasefig|6666666.22288.peg.2670 Microbial collagenase, secreted (EC 3.4.24.3)fig|6666666.22288.peg.3292 Microbial collagenase, secreted (EC 3.4.24.3)fig|6666666.22288.peg.3131 D-alanyl-D-alanine carboxypeptidase (EC3.4.16.4)

TABLE 3 Chitinases CDS ID Function fig|6666666.22288.peg.75N-acetylglucosamine-regulated outer membrane porinfig|6666666.22288.peg.893 Chitosanase precursor (EC 3.2.1.132)fig|6666666.22288.peg.1535 Beta-hexosaminidase (EC 3.2.1.52)fig|6666666.22288.peg.2867 Chitooligosaccharide deacetylase (EC 3.5.1.-)fig|6666666.22288.peg.2995 Chitinase (EC 3.2.1.14)fig|6666666.22288.peg.3355 Chitodextrinase precursor (EC 3.2.1.14)fig|6666666.22288.peg.4392 Chitinase (EC 3.2.1.14)fig|6666666.22288.peg.2782 Endoglucanase precursor (EC 3.2.1.4)

TABLE 4 Lipases, phospholipases and esterases CDS ID Functionfig|6666666.22288.peg.1665 Esterase/lipase fig|6666666.22288.peg.1695Lipase/acylhydrolase, putative fig|6666666.22288.peg.2171 Lipaseprecursor (EC 3.1.1.3) fig|6666666.22288.peg.2172 Lipase chaperone

Table 5 provides examples of C. subtsugae ORFs encoding secretedproteins with homology to various insect toxins.

TABLE 5 Toxins CDS ID Function fig|6666666.22288.peg.1582Channel-forming transporter/cytolysins activator of TpsB familyfig|6666666.22288.peg.1948 Channel-forming transporter/cytolysinsactivator of TpsB family fig|6666666.22288.peg.341 Probable thermolabilehemolysin fig|6666666.22288.peg.343 Phospholipase/lecithinase/hemolysinfig|6666666.22288.peg.670 21 kDa hemolysin precursor

Table 6 provides examples of C. subtsugae ORFs encoding potentiallysecreted proteins with effects on insect metabolism.

TABLE 6 Genes encoding proteins involved in iron acquisition andtransport CDS ID Function fig|6666666.22288.peg.541 Periplasmic proteinp19 involved in high-affinity Fe2+ transport fig|6666666.22288.peg.1533TonB-dependent receptor; Outer membrane receptor for ferrienterochelinand colicins fig|6666666.22288.peg.1540 Ferric iron ABC transporter,iron-binding protein fig|6666666.22288.peg.1690 ABC transporter(iron.B12.siderophore.hemin), periplasmic fig|6666666.22288.peg.1735ABC-type Fe3+ transport system, periplasmic componentfig|6666666.22288.peg.3202 Iron(III)-binding periplasmic proteinSfuA/Thiamin ABC transporter, substrate-bindingfig|6666666.22288.peg.3933 TonB-dependent hemin , ferrichrome receptorfig|6666666.22288.peg.3935 Periplasmic hemin-binding protein

Table 7 provides examples of C. subtsugae ORFs encoding potentiallysecreted proteins with effects on plant growth promotion

TABLE 7 CDS ID Function fig|6666666.22288.peg.1092 Polyamine Metabolismfig|6666666.22288.peg.1500 Arginine and Ornithine Degradation, PolyamineMetabolism fig|6666666.22288.peg.1984 GABA and putrescine metabolismfrom cluters, Polyamine Metabolism fig|6666666.22288.peg.1987 Putrescineutilization pathways fig|6666666.22288.peg.3123 Arginine and OrnithineDegradation fig|6666666.22288.peg.4138 Polyamine Metabolismfig|6666666.22288.peg.4415 Polyamine Metabolism

Table 8 provides an example of a C. subtsugae ORF encoding a secretedprotein involved in degradation of organic phosphate. Such proteins areuseful, for example, for bioremediation.

TABLE 8 CDS ID Function fig|6666666.22288.peg.1492 Methyl pamthionhydrolase( EC:3.5.- )

Genes Involved in Sysnthesis of Pili and Fimbriae

Table 9 provides examples of C. subtsugae ORFs encoding proteins withpossible involvement in host interactions, in particular, biogenesis ofpili and fimbriae. Some of these proteins contain a signal peptide (asindicated in the right-most column of the table) and are thereforelikely to be secreted. Others, which do not contain a signal sequence,may be intracellular or transmembrane proteins.

TABLE 9 Fimbrial and Type IV Pilus Genes CDS ID Function Signal peptidefig|6666666.22288.peg.520 Type IV pilus biogenesis protein PilQ Yesfig|6666666.22288.peg.1297 Fimbrial subunit protein Yesfig|6666666.22288.peg.3157 Type IV fimbrial biogenesis protein PilY1 Yesfig|6666666.22288.peg.488 Type IV fimbrial biogenesis protein FimT Nofig|6666666.22288.peg.489 Type IV pilus biogenesis protein PilE Nofig|6666666.22288.peg.490 Type IV fimbrial biogenesis protein PilY1 Nofig|6666666.22288.peg.491 Type IV fimbrial biogenesis protein PilX Nofig|6666666.22288.peg.492 Type IV fimbrial biogenesis protein PilW Nofig|6666666.22288.peg.493 Type IV fimbrial biogenesis protein PilV Nofig|6666666.22288.peg.519 Type IV pilus biogenesis protein PilP No

Transmembrane Proteins

C. subtsugae sequences were examined for the presence of a transmembranedomain, indicative of proteins that are displayed on the cell surface.C. subtsugae proteins containing a transmembrane domain are disclosed inthis section.

Table 10 provides examples of C. subtsugae ORFs encoding transmembranetransporter proteins.

TABLE 10 Transmembrane Transporters ID Protein fig|6666666.22288.peg.24Permease of the drug/metabolite transporter (DMT) superfamilyfig|6666666.22288.peg.77 Chitobiose ABC transport system, permeaseprotein 1 fig|6666666.22288.peg.78 probable ABC transporter sugarpermease fig|6666666.22288.peg.110 Permease of the drug/metabolitetransporter (DMT) superfamily fig|6666666.22288.peg.143 Benzoatetransport protein fig|6666666.22288.peg.185 probable transporttransmembrane protein fig|6666666.22288.peg.193 Ammonium transporterfig|6666666.22288.peg.249 Glutamate Aspartate transport system permeaseprotein GltK (TC 3.A.1.3.4) fig|6666666.22288.peg.250 GlutamateAspartate transport system permease protein GltJ (TC 3.A.1.3.4)fig|6666666.22288.peg.251 Glutamate Aspartate periplasmic bindingprotein precursor GltI (TC 3.A.1.3.4) fig|6666666.22288.peg.259 PutativeTolA protein fig|6666666.22288.peg.260 Tol biopolymer transport system,TolR protein fig|6666666.22288.peg.339 RND efflux system, inner membranetransporter CmeB fig|6666666.22288.peg.344 Arsenic efflux pump proteinfig|6666666.22288.peg.376 Biopolymer transport protein ExbD/TolRfig|6666666.22288.peg.382 COG0477: Permeases of the major facilitatorsuperfamily fig|6666666.22288.peg.393 RND efflux system, inner membranetransporter CmeB fig|6666666.22288.peg.400 RND efflux system, innermembrane transporter CmeB fig|6666666.22288.peg.404 ABC-type multidrugtransport system, permease component fig|6666666.22288.peg.411Uncharacterized ABC transporter, periplasmic component YrbDfig|6666666.22288.peg.412 Uncharacterized ABC transporter, permeasecomponent YrbE fig|6666666.22288.peg.416 Permeases of thedrug/metabolite transporter (DMT) superfamily fig|6666666.22288.peg.422Histidine permease YuiF fig|6666666.22288.peg.462 Permeases of the majorfacilitator superfamily fig|6666666.22288.peg.465 probable MFStransporter fig|6666666.22288.peg.496 major facilitator superfamilyMFS_1 fig|6666666.22288.peg.502 MFS transporterfig|6666666.22288.peg.512 Putative preQ0 transporterfig|6666666.22288.peg.528 Lipid A export ATP-binding/permease proteinMsbA (EC 3.6.3.25) fig|6666666.22288.peg.585 Permease of thedrug/metabolite transporter (DMT) superfamily fig|6666666.22288.peg.616major facilitator superfamily MFS_1 fig|6666666.22288.peg.622 Majorfacilitator superfamily fig|6666666.22288.peg.697 ABC superfamily(ATP-binding membrane) transport protein fig|6666666.22288.peg.703Twin-arginine translocation protein TatC fig|6666666.22288.peg.705Twin-arginine translocation protein TatA fig|6666666.22288.peg.748Manganese transport protein MntH fig|6666666.22288.peg.771 Permease ofthe drug/metabolite transporter (DMT) superfamilyfig|6666666.22288.peg.809 Histidine ABC transporter, permease proteinHisQ (TC 3.A.1.3.1) fig|6666666.22288.peg.810 Histidine ABC transporter,permease protein HisM (TC 3.A.1.3.1) fig|6666666.22288.peg.823 Aminoacid transporter fig|6666666.22288.peg.850 Acetate permease ActP(cation/acetate symporter) fig|6666666.22288.peg.862 TRAP-typeC4-dicarboxylate transport system, large permease componentfig|6666666.22288.peg.863 TRAP-type transport system, small permeasecomponent, predicted N- acetylneuraminate transporterfig|6666666.22288.peg.903 Sodium/glutamate symport proteinfig|6666666.22288.peg.910 Dipeptide transport system permease proteinDppC (TC 3.A.1.5.2) fig|6666666.22288.peg.911 Dipeptide transport systempermease protein DppB (TC 3.A.1.5.2) fig|6666666.22288.peg.912Dipeptide-binding ABC transporter, periplasmic substrate-bindingcomponent (TC 3.A.1.5.2) fig|6666666.22288.peg.965 Permeases of themajor facilitator superfamily fig|6666666.22288.peg.10224-hydroxybenzoate transporter fig|6666666.22288.peg.1080 Phosphatetransport system permease protein PstC (TC 3.A.1.7.1)fig|6666666.22288.peg.1081 Phosphate transport system permease proteinPstA (TC 3.A.1.7.1) fig|6666666.22288.peg.1084 Low-affinity inorganicphosphate transporter fig|6666666.22288.peg.1149 Ethanolamine permeasefig|6666666.22288.peg.1155 probable multidrug resistance proteinfig|6666666.22288.peg.1167 probable MFS transporterfig|6666666.22288.peg.1175 Di-/tripeptide transporterfig|6666666.22288.peg.1183 Lead, cadmium, zinc and merany transportingATPase (EC 3.6.3.3) (EC 3.6.3.5); Copper-translocating P-type ATPase (EC3.6.3.4) fig|6666666.22288.peg.1201 D-serine/D-alanine/glycinetransporter fig|6666666.22288.peg.1205 Permease of the drug/metabolitetransporter (DMT) superfamily fig|6666666.22288.peg.1221 Chromatetransport protein ChrA fig|6666666.22288.peg.1222 Chromate transportprotein ChrA fig|6666666.22288.peg.1232 Kef-type K+ transport systems,predicted NAD-binding component fig|6666666.22288.peg.1236Nitrate/nitrite transporter fig|6666666.22288.peg.1267 Magnesium andcobalt transport protein CorA fig|6666666.22288.peg.1275 Chromatetransport protein ChrA fig|6666666.22288.peg.1276 probable permease ofABC transporter fig|6666666.22288.peg.1282 Spermidine export proteinMdtI fig|6666666.22288.peg.1283 Spermidine export protein MdtJfig|6666666.22288.peg.1302 Permeases of the major facilitatorsuperfamily fig|6666666.22288.peg.1377 Protein-export membrane proteinSecF (TC 3.A.5.1.1) fig|6666666.22288.peg.1378 Protein-export membraneprotein SecD (TC 3.A.5.1.1) fig|6666666.22288.peg.1436 Permease of thedrug/metabolite transporter (DMT) superfamily fig|6666666.22288.peg.1460probable homoserine/homoserine lactone efflux proteinfig|6666666.22288.peg.1463 Serine transporter fig|6666666.22288.peg.1464Formate efflux transporter (TC 2.A.44 family) fig|6666666.22288.peg.1478Major facilitator superfamily precursor fig|6666666.22288.peg.1530Iron(III) dicitrate transport system permease protein FecD (TC3.A.1.4.1) fig|6666666.22288.peg.1539 Ferric iron ABC transporter,permease protein fig|6666666.22288.peg.1549 High-affinity branched-chainamino acid transport system permease protein LivH (TC 3.A.1.4.1)fig|6666666.22288.peg.1550 Branched-chain amino acid transport systempermease protein LivM (TC 3.A.1.4.1) fig|6666666.22288.peg.1567 Zinc ABCtransporter, inner membrane permease protein ZnuBfig|6666666.22288.peg.1609 Probable Co/Zn/Cd efflux system membranefusion protein fig|6666666.22288.peg.1610 RND multidrug effluxtransporter; Acriflavin resistance protein fig|6666666.22288.peg.1620Drug resistance transporter EmrB/QacA subfamilyfig|6666666.22288.peg.1643 Putative sulfate permeasefig|6666666.22288.peg.1645 Potassium-transporting ATPase A chain (EC3.6.3.12) (TC 3.A.3.7.1) fig|6666666.22288.peg.1646Potassium-transporting ATPase B chain (EC 3.6.3.12) (TC 3.A.3.7.1)fig|6666666.22288.peg.1647 Potassium-transporting ATPase C chain (EC3.6.3.12) (TC 3.A.3.7.1) fig|6666666.22288.peg.1675 HoxN/HupN/NixAfamily cobalt transporter fig|6666666.22288.peg.1691 ABC transporter(iron.B12.siderophore.hemin), permease componentfig|6666666.22288.peg.1723 Putative sodium-dependent transporterfig|6666666.22288.peg.1733 Thiamin ABC transporter, transmembranecomponent fig|6666666.22288.peg.1734 ABC transporter permease proteinfig|6666666.22288.peg.1785 Sulfate permease fig|6666666.22288.peg.1791Putative 10 TMS drug/metabolite exporter, DME family, DMT superfamilyfig|6666666.22288.peg.1827 Permease of the drug/metabolite transporter(DMT) superfamily fig|6666666.22288.peg.1845 putative hemin permeasefig|6666666.22288.peg.1869 Permeases of the major facilitatorsuperfamily fig|6666666.22288.peg.1876 Sulfate transport system permeaseprotein CysW fig|6666666.22288.peg.1877 Sulfate transport systempermease protein CysT fig|6666666.22288.peg.1905 Ferric iron ABCtransporter, permease protein fig|6666666.22288.peg.1925 Putativetransport protein fig|6666666.22288.peg.1936 Transporter, LysE familyfig|6666666.22288.peg.1939 Permease of the drug/metabolite transporter(DMT) superfamily fig|6666666.22288.peg.1960 Nucleoside permease NupCfig|6666666.22288.peg.1966 Transporter, LysE familyfig|6666666.22288.peg.1985 Putrescine transport system permease proteinPotH (TC 3.A.1.11.2) fig|6666666.22288.peg.1986 Putrescine transportsystem permease protein PotI (TC 3.A.1.11.2) fig|6666666.22288.peg.1995Periplasmic protein TonB, links inner and outer membranesfig|6666666.22288.peg.1997 Biopolymer transport protein ExbD/TolRfig|6666666.22288.peg.1998 Biopolymer transport protein ExbD/TolRfig|6666666.22288.peg.1999 Biopolymer transport protein ExbD/TolRfig|6666666.22288.peg.2000 Biopolymer transport protein ExbD/TolRfig|6666666.22288.peg.2003 Cobalt-zinc-cadmium resistance protein CzcA;Cation efflux system protein CusA fig|6666666.22288.peg.2006Oligopeptide transport system permease protein OppB (TC 3.A.1.5.1)fig|6666666.22288.peg.2007 Oligopeptide transport system permeaseprotein OppC (TC 3.A.1.5.1) fig|6666666.22288.peg.2095 Permease of thedrug/metabolite transporter (DMT) superfamily fig|6666666.22288.peg.2109L-lysine permease fig|6666666.22288.peg.2117 Permease of thedrug/metabolite transporter (DMT) superfamily fig|6666666.22288.peg.2126Biopolymer transport protein ExbD/TolR fig|6666666.22288.peg.2127Biopolymer transport protein ExbD/TolR fig|6666666.22288.peg.2132Oligopeptide transport system permease protein OppC (TC 3.A.1.5.1)fig|6666666.22288.peg.2158 TonB-dependent receptorfig|6666666.22288.peg.2164 Ferric enterobactin transport system permeaseprotein FepG (TC 3.A.1.14.2) @ ABC-type Fe3+-siderophore transportsystem, permease 2 component fig|6666666.22288.peg.2165 Ferricenterobactin transport system permease protein FepD (TC 3.A.1.14.2) @ABC-type Fe3+-siderophore transport system, permease componentfig|6666666.22288.peg.2166 Enterobactin exporter EntSfig|6666666.22288.peg.2169 RND efflux system, inner membrane transporterCmeB fig|6666666.22288.peg.2190 Dipeptide transport system permeaseprotein DppB (TC 3.A.1.5.2) fig|6666666.22288.peg.2191 Oligopeptidetransport system permease protein OppC (TC 3.A.1.5.1)fig|6666666.22288.peg.2200 Sodium/alanine symporter family proteinfig|6666666.22288.peg.2226 ABC transport system, permease component YbhRfig|6666666.22288.peg.2227 ABC transport system, permease component YbhSfig|6666666.22288.peg.2262 Lipid A export ATP-binding/permease proteinMsbA fig|6666666.22288.peg.2295 Malate Na(+) symporterfig|6666666.22288.peg.2312 Putative TEGT family carrier/transportprotein fig|6666666.22288.peg.2331 Cobalt-zinc-cadmium resistanceprotein CzcA; Cation efflux system protein CusAfig|6666666.22288.peg.2332 Cobalt-zinc-cadmium resistance protein CzcA;Cation efflux system protein CusA fig|6666666.22288.peg.2333 ProbableRND efflux membrane fusion protein fig|6666666.22288.peg.2335Lysine-specific permease fig|6666666.22288.peg.2427 Potassium effluxsystem KefA protein/Small-conductance mechanosensitive channelfig|6666666.22288.peg.2452 Predicted nucleoside ABC transporter,permease 1 component fig|6666666.22288.peg.2453 Predicted nucleoside ABCtransporter, permease 2 component fig|6666666.22288.peg.2483 Probablesodium-dependent transporter fig|6666666.22288.peg.2582Cytosine/purine/uracil/thiamine/allantoin permease family proteinfig|6666666.22288.peg.2586 Methionine ABC transporter permease proteinfig|6666666.22288.peg.2645 ABC-type sugar transport system, periplasmiccomponent fig|6666666.22288.peg.2673 TRANSPORTER, LysE familyfig|6666666.22288.peg.2719 Nucleoside permease NupCfig|6666666.22288.peg.2720 probable transporterfig|6666666.22288.peg.2741 FIG021862: membrane protein, exporterfig|6666666.22288.peg.2772 Oligopeptide transport system permeaseprotein OppC (TC 3.A.1.5.1) fig|6666666.22288.peg.2793 calcium/protonantiporter fig|6666666.22288.peg.2846 Nucleoside:H+ symporter:Majorfacilitator superfamily fig|6666666.22288.peg.2865 Permeases of themajor facilitator superfamily fig|6666666.22288.peg.2896 Taurinetransport system permease protein TauC fig|6666666.22288.peg.2932Chitobiose ABC transport system, permease protein 1fig|6666666.22288.peg.2933 N-Acetyl-D-glucosamine ABC transport system,permease protein 2 fig|6666666.22288.peg.2934 L-Proline/Glycine betainetransporter ProP fig|6666666.22288.peg.2936 probable Na/H+ antiporterfig|6666666.22288.peg.2945 Cystine ABC transporter, permease proteinfig|6666666.22288.peg.2975 Probable glucarate transporterfig|6666666.22288.peg.3057 Ribose ABC transport system, permease proteinRbsC (TC 3.A.1.2.1) fig|6666666.22288.peg.3061 Mg(2+) transport ATPaseprotein C fig|6666666.22288.peg.3065 L-lactate permeasefig|6666666.22288.peg.3101 Zinc ABC transporter, periplasmic-bindingprotein ZnuA fig|6666666.22288.peg.3102 Zinc ABC transporter, innermembrane permease protein ZnuB fig|6666666.22288.peg.3124 Histidine ABCtransporter, permease protein HisQ (TC 3.A.1.3.1)fig|6666666.22288.peg.3125 Histidine ABC transporter, permease proteinHisM (TC 3.A.1.3.1) fig|6666666.22288.peg.3144 Mg(2+) transport ATPase,P-type (EC 3.6.3.2) fig|6666666.22288.peg.3190 Sodium/bile acidsymporter family fig|6666666.22288.peg.3200 Thiamin ABC transporter,transmembrane component fig|6666666.22288.peg.3220 Long-chain fatty acidtransport protein fig|6666666.22288.peg.3275 L-lysine permeasefig|6666666.22288.peg.3277 L-lysine permease fig|6666666.22288.peg.3286Homolog of fucose/glucose/galactose permeases fig|6666666.22288.peg.3333Amino acid transporters fig|6666666.22288.peg.3374 Permease of thedrug/metabolite transporter (DMT) superfamily fig|6666666.22288.peg.3382Biopolymer transport protein ExbD/TolR fig|6666666.22288.peg.3451Permeases of the major facilitator superfamilyfig|6666666.22288.peg.3517 major facilitator family transporterfig|6666666.22288.peg.3531 Mg(2+) transport ATPase protein Cfig|6666666.22288.peg.3532 Manganese transport protein MntHfig|6666666.22288.peg.3534 Permease of the drug/metabolite transporter(DMT) superfamily fig|6666666.22288.peg.3609 Ferrous iron transportprotein B fig|6666666.22288.peg.3673 Uracil permeasefig|6666666.22288.peg.3700 probable sodium/alanine symporterfig|6666666.22288.peg.3704 Glycerol-3-phosphate ABC transporter,permease protein UgpE (TC 3.A.1.1.3) fig|6666666.22288.peg.3705Glycerol-3-phosphate ABC transporter, permease protein UgpA (TC3.A.1.1.3) fig|6666666.22288.peg.3777 Molybdenum transport systempermease protein ModB (TC 3.A.1.8.1) fig|6666666.22288.peg.3784 ABCtransporter, permease protein, putative fig|6666666.22288.peg.3787 majorfacilitator superfamily MFS_1 fig|6666666.22288.peg.3790 Transporterfig|6666666.22288.peg.3831 Arginine/ornithine antiporter ArcDfig|6666666.22288.peg.3887 Cobalt-zinc-cadmium resistance protein CzcA;Cation efflux system protein CusA fig|6666666.22288.peg.3888 ProbableCo/Zn/Cd efflux system membrane fusion proteinfig|6666666.22288.peg.3936 Hemin ABC transporter, permease proteinfig|6666666.22288.peg.3963 RND efflux transporterfig|6666666.22288.peg.4003 Ammonium transporterfig|6666666.22288.peg.4049 Amino acid ABC transporter, permease proteinfig|6666666.22288.peg.4068 ABC transporter, ATP-binding/permease proteinfig|6666666.22288.peg.4136 Spermidine Putrescine ABC transporterpermease component PotB (TC 3.A.1.11.1) fig|6666666.22288.peg.4137Spermidine Putrescine ABC transporter permease component potC(TC_3.A.1.11.1) fig|6666666.22288.peg.4180 POTASSIUM/PROTON ANTIPORTERROSB fig|6666666.22288.peg.4193 MFS permease fig|6666666.22288.peg.4233Osmoprotectant ABC transporter inner membrane protein YehWfig|6666666.22288.peg.4235 Putative ABC transport integral membranesubunit fig|6666666.22288.peg.4236 probable ABC transporterfig|6666666.22288.peg.4258 Sodium-dependent transporterfig|6666666.22288.peg.4300 Oligopeptide transport system permeaseprotein OppB (TC 3.A.1.5.1) fig|6666666.22288.peg.4301 Oligopeptidetransport system permease protein OppC (TC 3.A.1.5.1)fig|6666666.22288.peg.4326 Glycine betaine transporter OpuDfig|6666666.22288.peg.4337 major facilitator superfamily MFS_1fig|6666666.22288.peg.4345 ABC-type anion transport system, duplicatedpermease component fig|6666666.22288.peg.4373 probable TonB proteinfig|6666666.22288.peg.4380 Potassium-transporting ATPase A chain (EC3.6.3.12) (TC 3.A.3.7.1) fig|6666666.22288.peg.751 Kup system potassiumuptake protein fig|6666666.22288.peg.755 Putative preQ0 transporterfig|6666666.22288.peg.992 TonB-dependent receptorfig|6666666.22288.peg.1269 Lead, cadmium, zinc and merculy transportingATPase (EC 3.6.3.3) (EC 3.6.3.5); Copper-translocating P-type ATPase (EC3.6.3.4) fig|6666666.22288.peg.2902 Putative preQ0 transporterfig|6666666.22288.peg.3020 Sodium-dependent phosphate transporter

Table 11 provides examples of C. subtsugae ORFs encoding transmembraneproteases.

TABLE 11 Transmembrane Proteases fig|6666666.22288.peg.436 Peptidase M50fig|6666666.22288.peg.1909 Membrane carboxypeptidase (penicillin-bindingprotein) fig|6666666.22288.peg.2281 cell wall endopeptidase, familyM23/M37 fig|6666666.22288.peg.2516 probable Peptidasefig|6666666.22288.peg.2670 Microbial collagenase, secreted (EC 3.4.24.3)fig|6666666.22288.peg.4364 Peptidase M48, Ste24p precursorfig|6666666.22288.peg.2081 Signal peptidase I (EC 3.4.21.89)

Table 12 provides examples of C. subtsugae ORFs encoding transmembranetoxins.

TABLE 12 Transmembrane Toxins fig|6666666.22288.peg.308 probable colicinV secretion atp-binding protein fig|6666666.22288.peg.101 Hemolysins andrelated proteins containing CBS domains fig|6666666.22288.peg.670 21 kDahemolysin precursor fig|6666666.22288.peg.1187 Holin-like protein CidAfig|6666666.22288.peg.1949 Hemolysin fig|6666666.22288.peg.2123 probableporin protein fig|6666666.22288.peg.2602 Zonula occludens toxin-likefig|6666666.22288.peg.2638 Colicin V production proteinfig|6666666.22288.peg.2639 DedD protein fig|6666666.22288.peg.2877hemolysin secretion protein D fig|6666666.22288.peg.2878 cyclolysinsecretion ATP-binding protein fig|6666666.22288.peg.3656 Antiholin-likeprotein LrgA fig|6666666.22288.peg.3881 porin signal peptide proteinfig|6666666.22288.peg.307 HlyD family secretion protein

Table 13 provides examples of C. subtsugae ORFs encoding antibiotics andproteins involved in antibiotic resistance.

TABLE 13 fig|6666666.22288.peg.30 Beta-lactamase (EC 3.5.2.6)fig|6666666.22288.peg.48 rarD protein, chloamphenicol sensitivefig|6666666.22288.peg.540 Fosmidomycin resistance proteinfig|6666666.22288.peg.584 Polymyxin resistance protein ArnT,undecaprenyl phosphate-alpha-L-Ara4N transferase; Melittin resistanceprotein PqaB fig|6666666.22288.peg.587 Polymyxin resistance proteinArnC, glycosyl transferase (EC 2.4.-.-) fig|6666666.22288.peg.1176Polymyxin resistance protein ArnT, undecaprenyl phosphate-alpha-L-Ara4Ntransferase; Melittin resistance protein PqaB fig|6666666.22288.peg.1177Polymyxin resistance protein ArnC, glycosyl transferase (EC 2.4.-.-)fig|6666666.22288.peg.1509 Multiple antibiotic resistance protein marCfig|6666666.22288.peg.1736 Hydrogen cyanide synthase HcnC/Opine oxidasesubunit B fig|6666666.22288.peg.3072 Arsenical-resistance protein ACR3fig|6666666.22288.peg.3756 Multiple antibiotic resistance protein marCfig|6666666.22288.peg.4348 Undecaprenyl-phosphate N-acetylglucosaminyl1-phosphate transferase (EC 2.7.8.-)

Homologues

The present disclosure also provides methods of obtaining homologues ofthe fragments of the C. subtsugae genome disclosed herein, andhomologues of the proteins encoded by the ORFs disclosed herein.Specifically, by using the nucleotide and amino acid sequences disclosedherein as a probe or as primers, and techniques such as PCR cloning andcolony/plaque hybridization, one skilled in the art can obtain saidhomologues. Such homologues can be obtained from any organism; e.g.,other species of Chromobacterium or other bacteria.

Antibodies, Detection Methods, Kits

Also provided are antibodies which selectively bind a protein orpolypeptide fragment encoded by the C. subtsugae genome. Suchantibodies, in addition, can comprise a detectable label and/or beattached to a solid support. Such antibodies include both monoclonal andpolyclonal antibodies. Also provided are hybridomas which produce theabove-described monoclonal antibodies.

In additional embodiments, the present disclosure provides methods ofidentifying test samples derived from cells that express one or more ofthe ORFs disclosed herein, or homologues thereof. Such methods compriseincubating a test sample with one or more of the antibodies of thepresent disclosure, or one or more fragments of the C. subtsugae genome,under conditions which allow a skilled artisan to determine if thesample contains the ORF (or portion thereof) or product producedtherefrom.

In additional embodiments, kits are provided which contain the necessaryreagents to carry out the above-described assays. Specifically, providedherein is a compartmentalized kit designed to receive, in closeconfinement, one or more containers which comprises: (a) a firstcontainer comprising one of the antibodies, or one of the C. subtsugaegenome fragments of the present disclosure; and (b) one or more othercontainers comprising one or more of the following: wash reagents,reagents capable of detecting presence of bound antibodies or reagentscapable of detecting presence of hybridized nucleic acids.

Using the isolated proteins disclosed herein, the present disclosurefurther provides methods of obtaining and identifying agents capable ofbinding to a protein encoded by a C. subtsugae ORF. Specifically, suchagents include antibodies (described above), peptides, carbohydrates,pharmaceutical agents and the like. Such methods comprise the steps of:(a) contacting an agent with an isolated protein encoded by one of theORFs disclosed herein; and (b) determining whether the agent binds tosaid protein. Methods for detecting protein-protein binding arewell-known in the art and include, for example, filter-binding,immunoprecipitation, two-hybrid assays, gel retardation and reportersubunit complementation. See, for example, U.S. Pat. Nos. 5,503,977 and5,585,245; Fields et al. (1989) Nature 340:245-247; Bai et al. (1996)Meth. Enzymol. 273:331-347 and Luo et al. (1997) BioTechniques22:350-352.

Vectors

For embodiments in which a polypeptide is produced using recombinanttechniques, the methods can involve any suitable construct and anysuitable host cell, which can be a prokaryotic or eukaryotic cell (e.g.a bacterial host cell, a yeast host cell, a plant host cell, an insecthost cell, or a cultured mammalian host cell). Methods for introducinggenetic material into host cells are well-known in the art and include,for example, biolistics, transformation, electroporation, lipofection,conjugation, calcium phosphate co-precipitation and the like. The methodfor transfer can be selected so as to provide for stable expression ofthe introduced polypeptide-encoding nucleic acid. Thepolypeptide-encoding nucleic acid can be provided as an inheritableepisomal element (e.g., plasmid) or can be genomically integrated.

Viral vectors can also be used for cloning and expression of the nucleicacids disclosed herein. Exemplary plant viral vectors includecauliflower mosaic virus (CaMV), pea early browning virus (PEBV), beanpod mottle virus (BPMV), cucumber mosaic virus (CMV), apple latentspherical virus (ALSV), tobacco mosaic virus (TMV), potato virus X,brome mosaic virus (BMV) and barley stripe mosaic virus (BSMV).

Additional vectors can be used for expression of C. subtsugaepolypeptide sequences in non-plant organisms. These include prokaryoticcloning vectors (e.g., pBR322, pUC, bacteriophage lambda), fungalvectors (e.g., yeast 2-micron plasmid), insect cloning vectors (e.g.,baculovirus) and mammalian vectors (e.g., SV40).

Suitable vectors for transferring a polypeptide-encoding nucleic acidcan vary in composition. Integrative vectors can be conditionallyreplicative or suicide plasmids, bacteriophages, and the like. Theconstructs can include various elements, including for example,promoters, selectable genetic markers (e.g., genes conferring resistanceto antibiotics, for example, instance neomycin, G418, methotrexate,ampicillin kanamycin, erythromycin, chloramphenicol, or gentamycin),origins of replication (to promote replication in a host cell, e.g., abacterial host cell), and the like. The choice of vector depends upon avariety of factors such as the type of cell in which propagation isdesired and the purpose of propagation. Certain vectors are useful foramplifying and making large amounts of the desired DNA sequence. Othervectors are suitable for expression of protein in cells. Still othervectors are suitable for transfer and expression in cells in a wholeanimal or plant. The choice of appropriate vector is well within theskill of the art. Many such vectors are available commercially.

The vector used can be an expression vector based on episomal plasmidscontaining selectable drug resistance markers and elements that providefor autonomous replication in different host cells. Vectors are amplydescribed in numerous publications well known to those in the art,including, e.g., Short Protocols in Molecular Biology, (1999) F.Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expressionof the nucleic acids encoding the subject polypeptide, may provide forpropagating the subject nucleic acids, or both.

Constructs can be prepared by, for example, inserting a polynucleotideof interest into a construct backbone, typically by means of DNA ligaseattachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination or site-specific recombination, or by one ormore amplification methods (e.g., PCR). Typically homologousrecombination is accomplished by attaching regions of homology to thevector on the flanks of the desired nucleotide sequence, whilesite-specific recombination can be accomplished through use of sequencesthat facilitate site-specific recombination (e.g., cre-lox, att sites,etc.). Nucleic acid containing such sequences can be added by, forexample, ligation of oligonucleotides, or by polymerase chain reactionusing primers comprising both the region of homology and a portion ofthe desired nucleotide sequence.

For expression of the polypeptide of interest, an expression cassettecan be employed. Thus, the present disclosure provides a recombinantexpression vector comprising a subject nucleic acid. The expressionvector can provide transcriptional and translational regulatorysequences, and can also provide for inducible or constitutiveexpression, wherein the coding region is operably placed under thetranscriptional control of a transcriptional initiation region (e.g., apromoter, enhancer), and transcriptional and translational terminationregions. These control regions may be native to the C. subtsugae genome,or can be derived from exogenous sources. As such, control regions fromexogenous sources can be considered heterologous elements that areoperably linked to the nucleic acid encoding the subject polypeptide. Ingeneral, the transcriptional and translational regulatory sequences caninclude, but are not limited to, promoter sequences, operator sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, polyadenylation sites andenhancer or activator sequences. Promoters can be either constitutive orinducible, and can be a strong constitutive promoter (e.g., T7 promoter,SP6 promoter, and the like).

Exemplary plant regulatory sequences, which can be used in therecombinant constructs disclosed herein, include constitutive promoterssuch as the CaMV 19S and 35S promoters and those from genes encodingactin or ubiquitin. Alternatively, regulated promoters such aschemically-regulated promoters (e.g., tetracycline-regulated) andwound-inducible promoters (expressed at wound sites and at sites ofphytopathogenic infection) can also be used. In additional embodiments,promoters can be tissue-specific (e.g., specifying expression in roots,leaves, flowers, inflorescences) and/or temporally regulated (e.g.,specifying expression in seedlings).

Additional promoters for use in plant cells have been described. See,for example, Stanford et al. (1989) Mol. Gen. Genet. 215: 200-208; Xu etal. (1993) Plant Molec. Biol. 22: 573-588; Logemann et al. (1989) PlantCell 1: 151-158; Rohrmeier & Lehle (1993) Plant Molec. Biol. 22:783-792; Firek et al. (1993) Plant Molec. Biol. 22: 129-142 and Warneret al. (1993) Plant J. 3: 191-201.

Consensus plant translation initiation sequences (i.e., ribosome-bindingsites) have been described by Joshi (1987) Nucleic Acids Res.15:6643-6653 and in the Clontech Catalogue 1993/1994, page 210.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding proteins of interest. A selectable marker operativein the expression host can be present to facilitate selection of cellscontaining the vector. In addition, the expression construct can includeadditional elements. For example, the expression vector can have one ortwo replication systems, thus allowing it to be maintained, for example,in plant or insect cells for expression and in a prokaryotic host forcloning and amplification. In addition, the expression construct cancontain a selectable marker gene to allow the selection of transformedhost cells. Selection genes are well-known in the art and vary dependingon the host cell used.

Expression vectors provided herein contain the aforementioned nucleicacids and/or polynucleotides. Such expression vectors can containpromoters (e.g., T7 promoter, T3 promoter, SP6 promoter, E. coli RNApolymerase promoter, lac promoter and its derivatives, tac promoter, trppromoter, the arabinose-inducible P_(BAD) promoter, theL-rhamnose-inducible rhaP_(BAD) promoter, bacteriophage lambda promoters(e.g, P_(L)), CMV promoter, SV40 promoter, PGK promoter, EF-1alphapromoter), operators, transcription termination signals (e.g., SV40termination signal), splice sites (e.g., SV40 splice sites, beta-globinsplice site), ribosome binding sites, signal sequences (e.g.,immunoglobulin kappa signal sequence), epitopes tags (e.g., myc, FLAG),purification tags (e.g., His₆), replication origins and drug selectionmarkers. Linker sequences, encoding linker amino acids and/or comprisingrestriction enzyme recognition sites, or any other type of linkersequence, can also be operably linked to the nucleic acid encoding thesubject polypeptide present in the vectors disclosed herein.

Cosmid libraries can be prepared by methods known in the art. See, forexample, Maniatis et al. Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Press, 2^(nd) edition, 1989 and Sambrook et al., 2001.Such a library can be used for sequence-based screening and for any typefunctional screening of cells, or of supernatants, whole cell broths,cell-free lysates, or extracts derived from the cells. High throughputbiological assays for herbicidal screening, enzymatic activities,anti-cancer activity, etc. are known in the art and described in theliterature. See also Examples 7-11 herein.

Host Cells

The present disclosure further contemplates recombinant host cellscontaining an exogenous polynucleotide. Said polynucleotide can compriseone or more fragments of the C. subtsugae genome as disclosed herein, orcan encode one or more of the polypeptides of the present disclosure.Host cells can be procaryotic (e.g., bacterial) or eucaryotic (e.g.,yeast, insect, mammalian). The host can also be a synthetic cell.

In certain embodiments, the host cell is a microorganism. Suitablemicroorganisms are those capable of colonizing plant tissue (e.g. root,stems, leaves, flowers, internally and on the surface), or therhizosphere, in such manner that they come in contact with insect pests.Some of the host microorganisms can also be capable of colonizing thegut of an insect pest, and be capable of being transmitted from oneinsect to another. Host microorganisms can also colonize the gut andbody surface of a plant pest. The host cell can also be used as amicrobial factory for the production of C. subtsugae proteins, or forproduction of one or more compounds produced by the activity of C.subtsugae proteins such as, for example, peptides, lipids, lipopeptides,glycoproteins, secondary metabolites, antibiotics and small organiccompounds.

Gram-negative microorganisms suitable for heterologous expressioninclude: Escherichia coli (e.g., E. coli K12, E. coli BL21), Pseudomonassp.(e.g. Pseudomonas fluorescens, Pseudomonas putida, Psuedomonasaurantiaca, Psuedomonas aureofaciens, Psuedomonas protegens),Enterobacter sp. (e.g. Enterobacter cloacae), and Serratia sp. ExemplaryE. coli strains include E. coli BL21 and E. coli K12 for routineexpression. Other E. coli strains, for more specialized purposes, arethose which display protease deficiency (BL21-B838) and those whichoverexpress membrane proteins such as the BL21 derivative DE3, C41 (DE3)and C43 (DE3).

Methods for high-level expression of heterologous proteins in E. coliare known and include (a) IPTG-induction methods, (b) auto-inductionmethods, and (c) high cell-density IPTG-induction methods. See, forexample, Sivashanmugam et al. (2009).

Gram-positive microorganisms suitable for heterologous expressioninclude Bacillus sp. (e.g., Bacillus megaterium, Bacillus subtilis,Bacillus cereus), and Streptomyces sp. One advantage of using Bacillusas an expression host is that membersof this genus produce spores, whichprovide formulations with better stability and longer shelf life.Expression systems based on Bacillus megaterium and Bacillus subtilisare commercially available from MoBiTec (Germany). Nucleotide sequencesof interest can be expressed in Bacillus megaterium using under thecontrol of the promoter of the xylose operon.

Fungal microorganisms suitable for heterologous expression includeTrichoderma sp., Gliocadium, Saccharomyces cerevisiae, and Pichiapastoris. Heterologous DNA can be introduced into filamentous fungi byprotoplast-mediated transformation using polyethylene glycol (PEG) or byelectroporation-based methods. Particle bombardment is another methodthat has been successfully used to transform fungal cells.

Methods and compositions for transformation of Saccharomyces cerevisiaeare well-known in the art. For example, a nucleic acid can be clonedinto a suitable vector (e.g., the YES vectors (Invitrogen, Carlsbad,Calif.), under the control of an inducible promoter such as GAL1, andthe CYC1 terminator, and expressed in Saccharomyces cerevisiae. Theresulting cells can be tested for the desired activity, or for proteinexpression.

Heterologous expression can also be conducted in other yeast species(Jeffries et al., 2010), such as Pichia pastoris, Hansenula polymorpha,Arxula adenivorans and Yarrowia lipolytica. Transformation of Pichiapastoris can be achieved with the use of a commercial kit, such as thePichiaPink Expression System (Invitrogen, Carlsbad, Calif.), the PichiaClassic Protein Expression System or the Pichia GlycoSwitch (forglycosylated proteins) (Research Corporation Technologies, Tucson,Ariz.). For transformation of the yeasts Pichia pastoris or Hansenula.polymorpha, electroporation can also be used.

In certain embodiments, non-pathogenic symbiotic bacteria, which areable to live and replicate within plant tissues (i.e., endophytes), ornon-pathogenic symbiotic bacteria, which are capable of colonizing thephyllosphere or the rhizosphere (i.e., epiphytes) are used. Suchbacteria include bacteria of the genera Agrobacterium, Alcaligenes,Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia,Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomycesand Xanthomonas.

Symbiotic fungi, such as Trichoderma and Gliocladium can also be used ashosts for propagation and/or expression of the sequences disclosedherein.

Formulations and Pesticidal Compositions

The present disclosure provides pesticidal (e.g., insecticidal)compositions and formulations comprising the nucleic acids andpolypeptides disclosed herein.

A “pest” is an organism (procaryotic, eucaryotic or Archael) thatincreases mortality and/or slows, stunts or otherwise alters the growthof a plant. Pests include, but are not limited to, nematodes, insects,fungi, bacteria, and viruses.

A “pesticide” as defined herein, is a substance derived from abiological product, or a chemical substance, that increases mortalityand/or inhibits the growth rate of plant pests. Pesticides include butare not limited to nematocides, insecticides, herbicides, plantfungicides, plant bactericides, and plant viricides.

A “biological pesticide” as defined herein is a microorganism withpesticidal properties.

A “pesticidal composition” is a formulation comprising a pesticide andoptionally one or more additional components. Additional componentsinclude, but are not limited to, solvents (e.g., amyl acetate, carbontetrachloride, ethylene dichloride; kerosene, xylene, pine oil, andothers listed in EPA list 4a and 4b etc.), carriers, (e.g., organicflour, Walnut shell flour, wood bark), pulverized mineral (sulfur,diatomite, tripolite, lime, gypsum talc, pyrophyllite), clay(attapulgite bentonites, kaolins, volcanic ash, and others listed in EPAlist 4a and 4b), stabilizers, emulsifiers (e.g., alkaline soaps, organicamines, sulfates of long chain alcohols and materials such as alginates,carbohydrates, gums, lipids and proteins, and others listed in EPA list4a and 4b), surfactants (e.g., those listed in EPA list 4a and 4b),anti-oxidants, sun screens, a second pesticide, either chemical orbiological (e.g., insecticide, nematicide, miticide, algaecide,fungicide, bactericide), an herbicide an/or an antibiotic.

A “carrier” as defined herein is an inert, organic or inorganicmaterial, with which the active ingredient is mixed or formulated tofacilitate its application to plant or other object to be treated, orits storage, transport and/or handling.

Pesticidal compositions as disclosed herein are useful for modulatingpest infestation in a plant. The term “modulate” as defined herein isused to mean to alter the amount of pest infestation or rate of spreadof pest infestation. Generally, such alteration is a lowering of thedegree and/or rate and/or spread of the infestation.

The term “pest infestation” as defined herein, is the presence of a pestin an amount that causes a harmful effect including a disease orinfection in a host population or emergence of an undesired weed in agrowth system. Exemplary plant pests include, but are not limited to,mites (e.g., Tetranychus urticae (Two-spotted spider mite)), fruit flies(e.g., Drosophila suzukii, Drosophila melanogaster), house flies (e.g.,Musca domestica), arachnids (e.g., Acari spp.), root maggots(Anthomyidae spp., e.g. Cabbage Root Maggots), aphids (e.g., Myzuspersicae (green peach aphid)), Triozidae spp. (e.g., potato psyllid(Bactericera cockerelli)), beetles (Tenebrionidae spp., e.g., litterbeetles (Alphitobius diaperinus)), grubs (e.g., white grub (Cyclocephalalurida), Southern Masked Chafer (Rhizotrogus majalis), Japanese beetle(Popillia japonica) larvae, black vine weevil (Otiorhyncus sulcatus)larvae, Oriental beetle (Anomala orientalis) larvae, scarabs (e.g.,Scarabaeidae spp.), nematodes (e.g., Root-knot nematode (Meloidogynespp.)), fungi, bacteria, and various plant viruses, for example, Tobaccomosaic virus, Tomato spotted wilt virus, Tomato yellow leaf curl virus,Cucumber mosaic virus, Potato virus Y, Cauliflower mosaic virus, Africancassava mosaic virus, Plum pox virus, Brome mosaic virus, Potato virusX, Citrus tristeza virus, Barley yellow dwarf virus, Potato leaf rollvirus and Tomato bushy stunt virus.

Pesticidal compositions, as disclosed herein, can be used either forprophylactic or modulatory purposes. When provided prophylactically, thecompositions(s) are provided in advance of any symptoms of infestation.The prophylactic administration of the composition(s) serves to prevent,attenuate, or decrease the rate of onset of any subsequent infection orinfestation. When provided for modulatory purposes, the composition(s)are provided at (or shortly after) the onset of an indication ofinfection or infestation. Modulatory administration of the compound(s)serves to attenuate the pathological symptoms of the infection orinfestation and to increase the rate of recovery.

Additional methods can be employed to control the duration of action.Controlled-release can be achieved through the use of polymers tocomplex or absorb one or more of the components of the composition. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate compositionsas disclosed herein into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. Alternatively, instead of incorporating thesecompositions into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldelivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are known in the art.

Pesticidal compositions as disclosed herein, (e.g., pesticidal toxins)can be produced by expression of selected Chromobacterium substugaegenome sequences in heterologous hosts suitable for lab scale, pilotscale and manufacturing scale fermentation (e.g., E. coli, Psuedomonassp., yeast, etc.). Toxins can be produced by fermentation proceduresknown in the art using the heterologous host and formulated directly, orafter extraction and purification of the toxin from the fermentationbroth. The formulation can include live cells or non-viable cells.

The pesticidal compositions disclosed herein can be formulated in anymanner. Non-limiting formulation examples include, but are not limitedto, emulsifiable concentrates (EC), wettable powders (WP), solubleliquids (SL), aerosols, ultra-low volume concentrate solutions (ULV),soluble powders (SP), microencapsulates, water dispersed granules,flowables (FL), microemulsions (ME), nano-emulsions (NE), etc. In any ofthe formulations described herein, the percentage of the activeingredient is within a range of 0.01% to 99.99%. Detailed description ofpesticide formulations can be found in the Kirk-Othmer Encyclopedia ofChemical Technology.; Knowles, A. 2005. New Developments in CropProtection Product Formulation, Agrow Reports, London, UK; Valkenburg,W.van (ed.) 1973, Pesticide Formulation, Marcel Dekker, New York, USA;Knowles, D.A. (ed.) 1998, Chemistry and Technology of AgrochemicalFormulations, Kluwer Academic Publishers, Dordrecht, the Netherlands.

Powder and Dust Formulations

These are simple formulations that usually contain 0.1-25% of the activeingredient. However, higher concentrations of active ingredient can beused depending on the potency and particular application. The pesticidetoxin is mixed with a solid carrier, preferably of small particle size.Solid carriers can include: silicate clays (e.g., attapulgite,bentonites, volcanic ash, montmorillionite, kaolin, talc, diatomites,etc.), carbonates (e.g., calcite, dolomite, etc), synthetics(precipitated silica, fumed silica, etc.), ground botanicals (e.g., corncob grits, rice hulls, coconut shells, etc.), organic flour (e.g.,Walnut shell flour, wood bark, etc.) or pulverized mineral (e.g.,Sulphur, diatomite, tripolite, lime, gypsum talc, pyrophyllite, etc.).The inert ingredients used in dust formulations can also come from thoselisted in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations. Small particle size can be achieved by mixingthe active ingredient with the carrier and pulverizing in a mill. Dustsare defined as having a particle size less than 100 microns; and withincrease in particle size the toxicity of the formulation decreases. Inthe selection of a dust formulation its compatibility, fineness, bulkdensity, flow ability, abrasiveness, absorbability, specific gravity andcost should be taken into consideration. Exemplary dust formulations areprovided in Table 14.

TABLE 14 Formulation components Formulation A Formulation B FormulationC Formulation D Active ingredient 0.65 5 10 25 Talc 50 90 Kaolin orother 49.35 95 75 clay

A dust formulation can also be prepared from a dust concentrate (e.g.,40% active ingredient, 5% stabilizer, 20% silica, 35% magnesiumcarbonate) added at 1-10% to a 1:1 organic filler/talc combination.

The dust formulation is used as a contact powder (CP) or tracking powder(TP) against crawling insects.

A dust formulation with high flowability can be applied by pneumaticequipments in greenhouses.

Granular and Pellet Formulations

The pesticidal toxin is applied in liquid form to coarse particles ofporous material (e.g., clay, walnut shells, vermiculite, diatomaceousearth, corn cobs, attapulgite, montmorillioinite, kaolin, talc,diatomites, calcite, dolomite, silicas, rice hulls, coconut shells,etc.). The granules or pellets can be water dispersible, and can beformed by extrusion (for pesticidal actives with low water solubility),agglomeration or spray drying. Granules can also be coated orimpregnated with a solvent-based solution of the pesticidal toxin. Thecarrier particles can be selected from those listed in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations. The active ingredient can be absorbed by thecarrier material or coated on the surface of the granule. Particle sizecan vary from 250 to 1250 microns (0.25 mm to 2.38 mm) in diameter. Theformulations usually contain 2 to 10 percent concentration of thetoxicant. The granules are applied in water or whorls of plant or tosoil at the rate of 10 kg/ha. Granular formulations of systemicinsecticides are used for the control of sucking and soil pest byapplication to soil. Whorl application is done for the control of borerpests of crops such as sorghum, maize and sugarcane, etc. These types offormulations reduce drift and allow for slower release of the pesticidalcomposition.

Granular pesticides are most often used to apply chemicals to the soilto control weeds, fire ants, nematodes, and insects living in the soilor for absorption into plants through the roots. Granular formulationsare sometimes applied by airplane or helicopter to minimize drift or topenetrate dense vegetation. Once applied, granules release the activeingredient slowly. Some granules require soil moisture to release theactive ingredient. Granular formulations also are used to control larvalmosquitoes and other aquatic pests. Granules are used in agricultural,structural, ornamental, turf, aquatic, right-of-way, and public health(biting insect) pest control operations.

Application of granular formulations is common in pre-emergenceherbicides or as soil insecticides for direct application andincorporation into soil or other solid substrates where plants grow.Granules or pellets can also be applied in-furrow. Granules are commonlyused for application to water, such as in flooded rice paddies.

A typical granule formulation includes (%w/w) 1-40% active ingredient,1-2% stabilizer, 0-10% resin or polymer, 0-5% surfactant, 0-5% binderand is made up to 100% with the carrier material.

Wettable Powder Formulations

Wettable powder is a powdered formulation which yields a rather stablesuspension when diluted with water. It is formulated by blending thepesticidal agent with diluents such as attapulgite, a surface activeagent and auxiliary materials such as sodium salts of sulfo acids.Optionally stickers are added to improve retention on plants and othersurfaces. Wettable powders can be prepared by mixing the pesticidaltoxin (10-95%) with a solid carrier, plus 1-2% of a surface-active agentto improve suspensibility. The overall composition of the formulationincludes the active ingredient in solid form (5.0-75%), an anionicdispersant and an anionic or nonionic wetting agent.

A typical example of a wettable powder formulation includes 10-80%active ingredient, 1-2% wetting agents (e.g., benzene sulphonates,naphthalene sulphonates, aliphatic suplhosuccinates, aliphatic alcoholetoxylates, etc.), 2-5% dispersing agent (e.g., lignosulphonates,naphthalene sulphonate-formaldehyde condensates, etc.), and 0.1-1%antifoaming agent (e.g., isopar M (Exxon/Mobil)), made up to 100% withan inert filler or carrier (e.g., diatomaceous earth, silica, etc.).

Emulsifiable Concentrate (EC) Formulations

These are concentrated pesticide formulation containing an organicsolvent and a surface-active agent to facilitate emulsification withwater. When EC formulations are sprayed on plant parts, the solventevaporates quickly, leaving a deposit of toxin from which water alsoevaporates. Exemplary emulsifying agents in insecticide formulationsinclude alkaline soaps, organic amines, sulfates of long chain alcoholsand materials such as alginates, carbohydrates, gums, lipids andproteins. Emulsifying agents can be selected from those listed in EPAInert List 4a (www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) forconventional formulations and 4b(www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf) for organicformulations.

Solution Formulations

A solution formulation is a concentrated liquid pesticide formulationthat can be used directly, or require dilution in the case of a solubleconcentrate. Soluble concentrates and solutions are water- orsolvent-based mixtures with complete miscibility in water.

A typical example of a solution concentrate formulation includes 20-70%active ingredient, 5-15% wetting agent, 5-10% antifreeze, and is made upto 100% with water or a water miscible solvent.

Depending on the nature and stability of the pesticidal toxin, asolution formulation can optionally include thickeners, preservatives,antifoam, pH buffers, UV screens, etc.

Aerosol and Fumigant Formulations

In an insecticidal aerosol, the toxin is suspended as minute particleshaving sizes ranging from 0.1 to 50 microns in air as a fog or mist.This is achieved by burning the toxin or vaporizing it by heating. Thetoxicant dissolved in a liquefied gas, if released through small hole,may cause the toxicant particles to float in air with the rapidevaporation of the released gas.

A chemical compound, which is volatile at ambient temperatures andsufficiently toxic, is known as a fumigant. Fumigants generally enter aninsect via its tracheal system. Fumigants are used for the control ofinsect pests in storage bins, buildings and certain insects andnematodes in the soil. Most fumigants are liquids held in cans or tanksand often comprise mixtures of two or more gases. Alternatively,phosphine or hydrogen phosphide gas can be generated in the presence ofmoisture from a tablet made up of aluminium phosphide and ammoniumcarbonate. The advantage of using a fumigant is that sites that are noteasily accessible to other chemicals can be reached with fumigants, dueto the penetration and dispersal of the gas. Commonly used fumigants areEDCT, methyl bromide, aluminium phosphide and hydrocynic acid.

Formulation in Fertilizers Mixtures

A fertilizer mixture can be manufactured by addition of an insecticidalcomposition, as dislcosed herein, to a chemical fertilizer, or byspreading the composition directly on the fertilizer. Fertilizermixtures are applied at the regular fertilizing time and provide bothplant nutrients and control of soil insects. In an exemplary fertilizerformulation, urea (2% solution) is mixed with an insecticidalcomposition and sprayed for supply of nitrogen to the plant and forrealizing effective pest control.

Formulation as Poison Baits.

Poison baits consist of a base or carrier material attractive to thepest species and a chemical toxicant in relatively small quantities. Thepoison baits are used for the control of fruit flies, chewing insects,wireworms, white grubs in the soil, household pests, rats in the fieldand slugs. These formulations are useful for situations in which sprayapplication is difficult. A common base used in dry baits is wheat branmoistened with water and molasses. For the control of fruit suckingmoths fermenting sugar solution or molasses with a toxin is used.

Formulations for Seed Treatments

Seed treatments include application of a pesticidal composition,optionally in combination with other bioactive, antagonistic orsymbiotic agents, to the surface of a seed prior to sowing. Thepesticidal toxins, proteins, and/or compounds disclosed herein can beformulated for seed treatments in any of the following modes: drypowder, water slurriable powder, liquid solution, flowable concentrateor emulsion, emulsion, microcapsules, gel, or water dispersiblegranules; or can be applied to seeds by spraying on the seed beforeplanting.

In the case of a dry powder, the active ingredient is formulatedsimilarly to a wettable powder, but with the addition of a stickingagent, such as mineral oil, instead of a wetting agent. For example: onekg of purified talc powder (sterilized for 12 h), 15 g calciumcarbonate, and 10 g carboxymethyl cellulose are mixed under asepticconditions following the method described by Nandakumar et al (2001).Protein, nucleic acid suspensions or organisms expressing these aremixed in a 1:2.5 ratio (suspension to dry mix) and the product is shadedried to reduce moisture content to 20-35%.

The compositions can be in the form of a liquid, gel or solid.

A solid composition can be prepared by suspending a solid carrier in asolution of active ingredient(s) and drying the suspension under mildconditions, such as evaporation at room temperature or vacuumevaporation at 65° C. or lower. For liquid compositions, the activeingredient can be dissolved in a suitable carrier or solvent.

A composition can comprise gel-encapsulated active ingredient(s). Suchgel-encapsulated materials can be prepared by mixing a gel-forming agent(e.g., gelatin, cellulose, or lignin) with a composition comprising oneor more nucleic acids and/or polypeptides as disclosed herein, andoptionally a second pesticide or herbicide; and inducing gel formationof the agent.

The composition can additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)monolaurate. The concentration of surfactants can range between 0.1-35%of the total formulation, e.g., from 5-25%. The choice of dispersing andemulsifying agents, such as non-ionic, anionic, amphoteric and cationicdispersing and emulsifying agents, and the amount employed, isdetermined by the nature of the composition and the ability of the agentto facilitate the dispersion of the composition.

Formulations Comprising Microorganisms

Pesticidal compositions as set forth above can be combined with amicroorganism. The microorganism can be a plant growth promoter.Suitable microorganisms include, but are not limited to, Bacillus sp.(e.g., Bacillus firmus, Bacillus thuringiensis, Bacillus pumilus,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis),Paecilomyces sp. (P. lilacinus), Pasteuria sp. (P. penetrans),Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., Ampelomyces sp.,Pseudozyma sp., Streptomyces sp (S. bikiniensis, S. costaricanus, S.avermitilis), Burkholderia sp., Trichoderma sp., Gliocladium sp.,avermectin, Myrothecium sp., Paecilomyces spp., Sphingobacterium sp.,Arthrobotrys sp., Chlorosplenium sp., Neobulgaria sp., Daldinia sp.,Aspergillus sp., Chaetomium sp., Lysobacter sp., Lachnum papyraceum,Verticillium suchlasporium, Arthrobotrys oligospora, Verticilliumchlamydosporium, Hirsutella rhossiliensis, Pochonia chlamydosporia,Pleurotus ostreatus, Omphalotus olearius, Lampteromyces japonicas,Brevudimonas sp., Muscodor sp., Photorhabdus sp., and Burkholderia sp.Agents obtained or derived from such microorganisms can also be used incombination with the pesticidal nucleic acids and polypeptides disclosedherein.

Formulations Comprising Second Pesticides

Pesticidal compositions as set forth above can be combined with a asecond pesticide (e.g., nematocide, fungicide, insecticide, algaecide,miticide, or bactericide). Such an agent can be a natural oil oroil-product having fungicidal, bactericidal, nematicidal, acaricidaland/or insecticidal activity (e.g., paraffinic oil, tea tree oil,lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil,pyrethram). Furthermore, the pesticide can be a single site anti-fungalagent which may include but is not limited to benzimidazole, ademethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine,triazole), morpholine, hydroxypyrimidine, anilinopyrimidine,phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide,carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatichydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin,acylamine, phthalimide, benzenoid (xylylalanine); a demethylationinhibitor selected from the group consisting of imidazole, piperazine,pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole,propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole,fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil,an anthranilic diamide (e.g., chlorantranilipole) and a quinone outsideinhibitor (e.g., strobilurin). The strobilurin may include but is notlimited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). Theanti-fungal agent can also be derived from a Reynoutria extract.

The fungicide can also be a multi-site non-inorganic, chemical fungicideselected from the group consisting of chloronitrile, quinoxaline,sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios,phenylpyridin-amine, and cyano-acetamide oxime.

The composition can, as noted above, further comprise an insecticide.The insecticide can include but is not limited to avermectin, Bt (e.g.,Bacillus thuringiensis var. kurstaki), neem oil, spinosads, Burkholderiasp. (e.g., as set forth in WO2011/106491), entomopathogenic fungi such aBeauveria bassiana and chemical insecticides including but not limitedto organochlorine compounds, organophosphorous compounds, carbamates,pyrethroids, pyrethrins and neonicotinoids.

As noted above, the composition may further comprise a nematocide. Thisnematocide may include, but is not limited to, avermectin, microbialproducts such as Biome (Bacillus firmus), Pasteuria spp and organicproducts such as saponins.

Methods for Modulating Pest Infestation

Thus, according to the present disclosure, methods for modulating pestinfestation in a plant are provided. The methods comprise application toa plant, or to the soil or substrate in which the plant is growing, of apesticidal composition comprising a nucleic acid as disclosed herein;i.e., any of SEQ ID NOs: 1-4533, or any of the nucleic acids ofembodiments 1-7, 15-17 and 49-52, or any of the vectors of embodiments 8and 9.

Additional methods for modulating pest infestation in a plant compriseapplication, to a plant, or to the soil or substrate in which the plantis growing, of a pesticidal composition comprising a polypeptide asdisclosed herein; i.e., any of SEQ ID NOs: 4534-8960, or any of thepolypeptides of embodiments 10-14 and 53.

When used as biological insect control agents, insecticidal toxinsencoded by the C. subtsugae genome can be produced by expression of a C.subtsugae nucleotide sequence in a heterologous host cell capable ofexpressing the nucleotide sequences. In one embodiment, one or more C.subtsugae nucleotide sequences are inserted into an appropriateexpression cassette comprising, e.g., a promoter and a transcriptionaltermination signal. Expression of the nucleotide sequence(s) can beconstitutive or inducible, depending on the promoter and/or externalstimuli. In certain embodiments, the cell in which the toxin isexpressed is a microorganism, such as a virus, a bacterium, or a fungus.

In certain embodiments, a virus, such as a baculovirus, is engineered tocontain a C. subtsugae nucleotide sequence in its genome. Such arecombinant virus can express large amounts of, e.g., an insecticidaltoxin after infection of appropriate eukaryotic cells that are suitablefor virus replication and expression of the nucleotide sequence. Theinsecticidal toxin thus produced is used as an insecticidal agent.Alternatively, baculoviruses engineered to include the nucleotidesequence are used to infect insects in vivo and kill them, either byexpression of the insecticidal toxin or by a combination of viralinfection and expression of the insecticidal toxin.

Thus, the compositions set forth above, comprising C. subtsugae nucleicacids and polypeptides, can be used as pesticides. In particular, thecompositions as set forth above can be used as, for example,insecticides and nematicides, alone or in combination with one or moresecond pesticidal substances as set forth herein.

Specifically, nematodes that may be controlled using the method setforth above include but are not limited to parasitic nematodes such asroot-knot, cyst, and lesion nematodes, including but not limited to seedgall nematodes (Afrina wevelli), bentgrass nematodes (Anguina agrostis),shoot gall nematodes (Anguina spp.), seed gall nematodes (Anguina spp.,A. amsinckiae, A. balsamophila; A. tritici), fescue leaf gall nematodes(A. graminis), ear-cockle (or wheat gall) nematodes (Anguina tritici),bud and leaf (or foliar) nematodes (Aphelenchoides spp., A. subtenuis),begonia leaf (or fern, or spring crimp, or strawberry foliar, orstrawberry nematodes, or summer dwarf) nematodes (A. fragariae), fernnematodes (A. olesistus), rice nematodes (A. oryzae), currant nematodes(A. ribes), black currant (or chrysanthemum) nematodes (A. ritzemabosi),chrysanthemum foliar or leaf nematodes (A. ritzemabosi), rice white-tip(or spring dwarf, or strawberry bud) nematodes (A. besseyi),fungus-feeding (mushroom) nematodes (Aphelenchoides composticola),Atalodera spp. (Atalodera lonicerae, Atalodera ucri), spine nematodes(Bakernema variabile), sting nematodes (Belonolaimus spp., B. gracilis,B. longicaudatus), pine wood nematodes (Bursaphalenchus spp., B.xylophilus, B. mucronatus), sessile nematodes (Cacopaurus spp., C.epacris, C. pestis), amaranth cyst nematodes (Cactodera amaranthi),birch cyst nematodes (C. betulae), cactus cyst nematodes (C. cacti),estonian cyst nematodes (C. estonica), Thorne's cyst nematodes (C.thornei), knotweed cyst nematodes (C. weissi), ring nematodes (Criconemaspp.), spine nematodes (Criconema spp., C. civellae, C. decalineatum, C.spinalineatum), ring nematodes (Criconemella axeste, C. curvata, C.macrodora, C. parva), ring nematodes (Criconemoides spp., C. citri, C.simile), spine nematodes (Crossonema fimbriatum), eucalypt cystoidnematodes (Cryphodera eucalypti), bud, stem and bulb nematodes(Ditylenchus spp., D. angustus, D. dipsaci, D. destructor, D.intermedius), Mushroom spawn nematodes (D. myceliophagus), awl nematodes(Dolichodorus spp., D. heterocephalus, D. heterocephalous), spearnematodes (Dorylaimus spp.), stunt nematodes (Geocenamus superbus), cystnematodes (Globodera spp.), yarrow cyst nematodes (G. achilleae),milfoil cyst nematodes (G. millefolii), apple cyst nematodes (G. mali),white cyst potato nematodes (G. pallida), golden nematodes (G.rostochiensis), tobacco cyst nematodes (G. tabacum), Osborne's cystnematodes (G. tabacum solanacearum), horsenettle cyst nematodes (G.tabacum virginiae), pin nematodes (Gracilacus spp., G. idalimus), spiralnematodes (Helicotylenchus spp., H. africanus, H. digonicus, H.dihystera, H. erythrinae, H. multicinctus, H. paragirus, H.pseudorobustus, H. solani, H. spicaudatus), sheathoid nematodes(Hemicriconemoides spp., H. biformis, H. californianus, H. chitwoodi, H.floridensis, H. wessoni),sheath nematodes (Hemicycliophora spp., H.arenaria, H. biosphaera, H. megalodiscus, H. parvana, H. poranga, H.sheri, H. similis, H. striatula), cyst nematodes (Heterodera spp.),almond cyst nematodes (H. amygdali), oat (or cereal) cyst nematodes (H.avenae), Cajanus (or pigeon pea) cyst nematodes (H. cajani),bermudagrass (or heart-shaped, or Valentine) cyst nematodes (H.cardiolata), carrot cyst nematodes (H. carotae), cabbage cyst nematodesor brassica root eelworm (H. cruciferae), nutgrass (or sedge) cystnematodes (H. cyperi), Japanese cyst nematodes (H. elachista), fig (orficus, or rubber) cyst nematodes (H. fici), galeopsis cyst nematodes (H.galeopsidis), soybean cyst nematodes (H. glycines), alfalfa root (or peacyst) nematodes (H. goettingiana), buckwheat cyst nematodes (H.graduni), barley cyst nematodes (H. hordecalis), hop cyst nematodes (H.humuli), Mediterranean cereal (or wheat) cyst nematodes (H. latipons),lespedeza cyst nematodes (H. lespedezae), Kansas cyst nematodes (H.longicolla), cereals root eelworm or oat cyst nematodes (H. major),grass cyst nematodes (H. mani), lucerne cyst nematodes (H. medicaginis),cyperus (or motha) cyst nematodes (Heterodera mothi), rice cystnematodes (H. oryzae), Amu-Darya (or camel thorn cyst) nematodes (H.oxiana), dock cyst nematodes (H. rosii), rumex cyst nemtodes (H.rumicis), sugar beet cyst nematodes (H. schachtii), willow cystnematodes (H. salixophila), knawel cyst nematodes (H. scleranthii),sowthistle cyst nematodes (H. sonchophila), tadzhik cyst nematodes (H.tadshikistanica), turkmen cyst nematodes (H. turcomanica), clover cystnematodes (H. trifolii), nettle cyst nematodes (H. urticae), ustinovcyst nematodes (H. ustinovi), cowpea cyst nematodes (H. vigni), corncyst nematodes (H. zeae), rice root nematodes (Hirschmanniella spp., H.belli, H. caudacrena, H. gracilis, H.oryzae), lance nematodes(Hoplolaimus spp.), Columbia nematodes (H. columbus), Cobb's lancenematodes (H. galeatus), crown-headed lance nematodes (H.tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis),needle nematodes (Longidorus spp., L. africanus, L. sylphus), ringnematodes (Macroposthonia(=Mesocriconema)xenoplax), cystoid nematodes(Meloidodera spp.), pine cystoid nematodes (M floridensis), tadzhikcystoid nematodes (M. tadshikistanica), cystoid body nematodes(Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M.conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogynespp., M acronea, M. arenaria, M.artiellia, M. brevicauda, M. camelliae,M. carolinensis, M. chitwoodi, M. exigua, M. graminicola, M. hapla, M.hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M.javanica, M. kikuyuensis, M. konaensis, M. mali, M. microtyla, M. naasi,M. ovalis, M. platani, M. querciana, M. sasseri, M. tadshikistanica, M.thamesi), knapweed nematodes (Mesoanguina picridis), Douglas firnematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbusaberrans, N. batatiformis, N. dorsalis), sour paste nematodes(Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes(Paralongidorus microlaimus), spiral nematodes (Pararotylenchus spp.),stubby-root nematodes (Paratrichodorus allius, P. minor, P. porosus, P.renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P.bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P.holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P.similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P.brachyurus, P. coffeae, P. convallarias, P. crenatus, P.flakkensis, P.goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P.neglectus, P.penetrans, P. pratensis, P. scribneri, P. thornei, P.vulnus, P. zeae), stem gall nematodes (Pterotylenchus cecidogenus),grass cyst nematodes (Punctodera punctate), stunt nematodes(Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholusspp.), banana-root nematodes (R. similis), rice-root nematodes (R.oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchuscocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R.parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R.christiei, R. robustus), Thorne's lance nematodes (R. uniformis),Sarisodera hydrophylla, spiral nematodes (Scutellonema spp., S.blaberum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S.conicephalum), grass root-gall nematodes (Subanguina radicicola), roundcystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes(Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T.primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (orstylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T.aspericutis, T. claytoni, T.ebriensis, T. elegans, T. golden,T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T.oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulussemipenetrans), and dagger nematodes (Xiphinema spp., X. americanum, X.bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X.diversicaudatum X. index, X. insigne, X. nigeriense, X. radicicola, X.setariae, X. vulgarae, X. vuittenezi).

Phytopathogenic insects controlled by the methods set forth aboveinclude but are not limited to non-Culicidae larvae insects from theorder (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp.,Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp.,Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp.,Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp.,Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasiaspp., Cochylis spp., Coleophora spp., Crocidolomia binotalis,Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsiscastanea, Earias spp., Ephestia spp., Eucosma spp., Eupoeciliaambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedyanubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferialycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesiabotrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestrabrassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammenespp., Pandemis spp., Panolis flammea, Pectinophora gossypiella,Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella,Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp.,Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp.,Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example,Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp-, Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp.; (c) Orthoptera, for example, Blatta spp., Blattellaspp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplanetaspp. and Schistocerca spp.; (d) Isoptera, for example, Reticulitermesspp.; (e) Psocoptera, for example, Liposcelis spp.; Anoplura, forexample, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigusspp. and Phylloxera spp.; (g) Mallophaga, for example, Damalinea spp.and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniellaspp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaciand Scirtothrips aurantii; (i) Heteroptera, for example, Cimex spp.,Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp.,Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergellasingularis, Scotinophara spp. and Tniatoma spp.; (j) Homoptera, forexample, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri; (k) Hymenoptera, for example, Acromyrmex, Atta spp.,Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampaspp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsisspp. and Vespa spp.; (l) Diptera, for example, Aedes spp., Antherigonasoccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp.,Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophilamelanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypodermaspp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp.,Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyiahyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxysspp., Tabanus spp., Tannia spp. and Tipula spp.; (m) Siphonaptera, forexample, Ceratophyllus spp. und Xenopsylla cheopis and (n) from theorder Thysanura, for example, Lepisma saccharina.

The pesticidal compositions disclosed herein may further be used forcontrolling crucifer flea beetles (Phyllotreta spp.), root maggots(Delia spp.), cabbage seedpod weevil (Ceutorhynchus spp.) and aphids inoil seed crops such as canola (rape), mustard seed, and hybrids thereof,and also rice and maize. In a particular embodiment, the insect is amember of the Spodoptera, more particularly, Spodoptera exigua, Myzuspersicae, Plutella xylostella or Euschistus sp.

Application of an effective pesticidal control amount of a pesticidalcomposition as disclosed herein is provided. Said pesticidal compositionis applied, alone or in combination with another pesticidal substance,in an effective pest control or pesticidal amount. An effective amountis defined as that quantity of pesticidal composition, alone or incombination with another pesticidal substance, that is sufficient toprevent or modulate pest infestation. The effective amount and rate canbe affected by pest species present, stage of pest growth, pestpopulation density, and environmental factors such as temperature, windvelocity, rain, time of day and seasonality. The amount that will bewithin an effective range in a particular instance can be determined bylaboratory or field tests.

Methods of Application

The pesticidal compositions disclosed herein, when used in methods formodulating pest infestation, can be applied using methods known in theart. Specifically, these compositions can be applied to plants or plantparts by spraying, dipping, application to the growth substrate (e.g.,soil) around the plant, application to the root zone, dipping rootsprior to planting, application to plants as a turf or a drench, throughirrigation, or as soil granules. Plants are to be understood as meaningin the present context all plants and plant populations such as desiredand undesired wild plants or crop plants (including naturally occurringcrop plants). Crop plants can be plants obtained by conventional plantbreeding and optimization methods, by biotechnological and geneticengineering methods or by combinations of these methods, includingtransgenic plants and plant cultivars protectable or not protectable byplant breeders' rights. Plant parts are to be understood as meaning allparts and organs of plants above and below the ground, such as shoot,leaf, flower and root, examples which may be mentioned being leaves,needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots,tubers and rhizomes. The plant parts also include harvested material,and vegetative and generative propagation material, for examplecuttings, tubers, rhizomes, off-shoots and seeds.

Application can be external, (e.g. by spraying, fogging or painting) orinternal (e.g., by injection, transfection or the use of an insectvector). When applied internally, the compositions can be intracellularor extracellular (e.g., present in the vascular system of the plant,present in the extracellular space).

Treatment of the plants and plant parts with the compositions set forthabove can be carried out directly or by allowing the compositions to acton a plant's surroundings, habitat or storage space by, for example,immersion, spraying, evaporation, fogging, scattering, painting on,injecting. In the case in which the composition is applied to a seed,the composition can be applied to the seed as one or more coats prior toplanting the seed using methods known in the art.

Pesticidal compositions as disclosed herein can also be applied toseeds; e.g., as a seed coating. Different adherents (“stickers”) can beused in the manufacture of seed coatings, including, for example, methylcellulose, alginate, carrageenan and polyvinyl alcohol. The adherent isdissolved in water to a percentage between 1-10% and stored at roomtemperature before application to the seeds. Seeds are soaked inadherent solution (3 ml/100 seeds) for 15 min, scooped out and mixedwith organic matter (1.5 g/100 seeds) in plastic bags and shakenvigorously. This process can also be automated using a seed coatingmachine.

For priming seeds with compositions as disclosed herein, seeds aresoaked in twice the seed volume of sterile distilled water containingbacterial/protein/nucleic acid suspensions or talc formulation (dryformulation) (4-10 g kg⁻¹ of seed, depending on seed size) and incubatedat 25±2° C. for 12-24 h. The suspension is then drained off and theseeds are dried under shade for 30 min and used for sowing.

The compositions can also be used as soil amendments, e.g., incombination with a carrier such as a talc formulation. Formulations forsoil amendment can also include clays, emulsifiers, surfactants andstabilizers, as are known in the art. For preparation of talc basedformulations, one kg of purified talc powder (sterilized for 12 h), 15 gcalcium carbonate, and 10 g carboxymethyl cellulose are mixed underaseptic conditions following the method described by Nandakumar et al.(2001). Protein, nucleic acid suspensions or organisms expressing theseare mixed in a 1:2.5 ratio (suspension to dry mix) and the product isshade-dried to reduce moisture content to 20-35%.

For soil amendment, formulations (e.g., talc formulations) can beapplied at rates between 2.5-10 Kg ha⁻¹ at sowing and/or at differenttimes after emergence, or both, depending on the crops.

The compositions disclosed herein can also be applied to soil usingmethods known in the art. See, for example, the USDA website atnaldc.nal.usda.gov/download/43874/pdf, accessed Feb. 20, 2013. Suchmethods include but are not limited to fumigation, drip irrigation orchemigation, broadcast application of granules or sprays, soilincorporation (e.g., application of granules), soil drenching, seedtreatment and dressing, and bare root dip.

Plant Transformation

The nucleic acids disclosed herein can be introduced into, andoptionally expressed in, plants, using any of a number of planttransformation techniques. Transformation of plants can be undertakenwith a single DNA species or multiple DNA species (i.e.,co-transformation).

In certain embodiments, a C. subtsugae protein or polypeptide (e.g., atoxin) is expressed in a plant and provides protection to the plant frominsect pests. For example, a nucleotide sequence as disclosed herein canbe inserted into an expression cassette, which can optionally be stablyintegrated into the chromosome of a plant. In certain embodiments, thenucleotide sequence is included in a non-pathogenic self-replicatingvirus. Plants transformed in accordance with the present disclosure canbe monocots or dicots and include but are not limited to, maize, wheat,barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear,quince, melon, plum, cherry ,apricot, strawberry, papaya, avocado,mango, banana, alfalfa, rice, potato, eggplant, peach, cotton, carrot,tobacco, sorghum, nectarine, sugar beet, sugarcane, sunflower, soybean,tomato, pineapple, grape, raspberry, blackberry, cucumber, Arabidopsis,and woody plants such as coniferous and deciduous trees.

Once the desired nucleotide sequence has been introduced into aparticular plant species, it can be propagated in that species, ortransferred to other varieties of the same species, particularlyincluding commercial varieties, using traditional breeding techniques.

DNA can be introduced into plant cells through the use of a number ofart-recognized methods. Those skilled in the art will appreciate thatthe choice of methods can depend on the type of plant targeted fortransformation. Suitable methods for transforming plant cells are asfollows.

Agrobacterium-Mediated Transformation

A major method of DNA transfer in plants is Agrobacterium mediatedtransformation. The natural living soil bacterium Agrobacteriumtumefaciens is capable of infecting a wide range of plant species,causing Crown Gall diseases. When A. tumefaciens infects a cell, ittransfers a copy of its T-DNA, which is a small section of DNA carriedon its Ti (Tumor Inducing) plasmid. The T-DNA is flanked by two(imperfect) 25 base pair repeats. Any DNA contained within these borderswill be transferred to the host cell. Zupan and Zambriski, 1995. TheT-DNA section on the Ti plasmid can be replaced by a transgene attachedto an appropriate regulatory sequence(s). Recombinant A. tumeficienscontaining a Ti plasmid comprising exogenous nucleotide sequences canthen be used to infect cultures of either regenerating cell orprotoplasts (i.e., wall-less spherical plant cells). Marker genes suchas those coding for antibiotic resistance can be included in the Tiplasmid construct, so that it is possible to select cells that have beentransformed by the bacterium. Cell-to-plant regeneration is carried outon the selected cells by standard methods. See, for example, Zupan andZambriski (1995) and Jones et al. (2005) Plant Methods.

Agrobacterium tumefaciens can used to transform many dicotyledonousplant species with relative ease. Hinchee et al., Biotechnology6:915-921 (1988). See also Ishida et al., Nature Biotechnology14:745-750 (June 1996) for a description of maize transformation.

Biolistic Delivery

This method, also known as “particle bombardment,” involves directly“shooting” a DNA molecule into the recipient plant tissue, using a “genegun.” Tungsten or gold beads (which are smaller than the plant cellsthemselves) are coated with the DNA of interest and fired through astopping screen, accelerated by Helium, into the plant tissue. Theparticles pass through the plant cells, leaving the DNA inside. Thismethod can be used on both monocotyledonous and dicotyledonous speciessuccessfully. Transformed tissue can be selected using marker genes suchas those encoding antibiotic resistance. Whole plants, containing a copyof the transgene in all cells, can be regenerated from the totipotenttransformed cells in culture (Nottingham, 1998), using devices availablefrom Agracetus, Inc. (Madison, Wis.) and Dupont, Inc. (Wilmington,Del.).

Methods for biolistic plant transformation are well-known in the art.See, for example, Sanford et al., U.S. Pat. No. 4,945,050; McCabe etal., Biotechnology 6.923-926 (1988); Weissinger et al., Annual RevGenet. 22-421-477 (1988); Sanford et al., Particulate Science andTechnology 5.27-37 (1987)(onion); Svab et al., Proc. Natl. Acad. Sci.USA 87-8526-8530 (1990) (tobacco chloroplast); Christou et al., PlantPhysiol 87,671-674 (1988)(soybean); McCabe et al., BioTechnology6.923-926 (1988)(soybean); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305-4309 (1988)(maize); Klein et al., BioTechnology 6,559-563 (1988)(maize); Klein et al., Plant Physiol. 91,440-444 (1988) (maize); Frommet al., BioTechnology 8:833-839 (1990); Gordon-Kamm et al., Plant Cell2: 603-618 (1990) (maize); Koziel et al., Biotechnology 11: 194-200(1993) (maize); Shimamoto et al., Nature 338: 274-277 (1989) (rice);Christou et al., Biotechnology 9: 957-962 (1991) (rice); Datta et al.,BioTechnology 8.736-740 (1990) (rice); European Patent Application EP 0332 581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology11: 1553-1558 (1993) (wheat); Weeks et al., Plant Physiol. 102:1077-1084(1993) (wheat); Wan et al., Plant Physiol. 104:37-48 (1994) (barley);Jahne et al., Theor. Appl. Genet. 89:525-533 (1994)(barley); Umbeck etal., BioTechnology 5:263-266 (1987) (cotton); Casas et al., Proc. Natl.Acad. Sci. USA 90:11212-11216 (December 1993) (sorghum); Somers et al.,BioTechnology 10:1589-1594 (December 1992) (oat); Torbert et al., PlantCell Reports 14:635-640 (1995) (oat); Weeks et al., Plant Physiol.102:1077-1084 (1993) (wheat); Chang et al., WO 94/13822 (wheat) andNehra et al., The Plant Journal 5:285-297 (1994) (wheat).

Methods for the introduction of recombinant DNA molecules into maize bymicroprojectile bombardment can be found in Koziel et al., Biotechnology11: 194-200(1993), Hill et al., Euphytica 85:119-123 (1995) and Kozielet al., Annals of the New York Academy of Sciences 792:164-171 (1996).

Protoplast Transformation and Other Methods

Another method for the introduction of nucleic acid molecules intoplants is the protoplast transformation method for maize as disclosed inEP 0 292 435. Additional delivery systems for gene transfer in plantsinclude electroporation (Riggs et al., Proc. Natl. Acad, Sci. USA83,5602-5606 (1986), microinjection (Crossway et al., BioTechniques4,320-334 (1986), silicon carbide-mediated DNA transfer, direct genetransfer (Paszkowski et al., EMBO J. 3.2717-2722 (1984); Hayashimoto etal., Plant Physiol 93.857-863 (1990)(rice).

Plastid Transformation

In another embodiment, a nucleotide sequence as disclosed herein isdirectly transformed into the genome of a plastid (e.g., chloroplast).Advantages of plastid transformation include the ability of plastids toexpress bacterial genes without substantial modification of thebacterial sequences, and the ability of plastids to express multipleopen reading frames under the control of a single promoter. Plastidtransformation technology is described in U.S. Pat. Nos. 5,451,513;5,545,817 and 5,545,818; in PCT application No. WO 95/16783, and inMcBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305.

The basic technique for chloroplast transformation involves introducingregions of cloned plastid DNA flanking a selectable marker, togetherwith the gene of interest, into a suitable target tissue using, e.g.,biolistics or protoplast transformation (e.g., calcium chloride or PEGmediated transformation). The 1 to 1.5 kb flanking regions, termedtargeting sequences, facilitate homologous recombination with theplastid genome and thus allow the replacement or modification ofspecific regions of the plastid genome. Initially, point mutations inthe chloroplast 16S rRNA and rpsl2 genes conferring resistance tospectinomycin and/or streptomycin were utilized as selectable markersfor transformation (Svab, Z. et al., (1990) Proc. Natl. Acad. Sci. USA87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45);resulting in the production of stable homoplasmic transformants at afrequency of approximately one per 100 bombardments of target leaves.The presence of cloning sites between these markers allowed creation ofa plastid targeting vector for introduction of foreign genes. Staub, J.M., and Maliga, P. (1993) EMBO J. 12: 601-606. Substantial increases intransformation frequency were obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial AADA gene encoding the spectinomycin-detoxifyingenzyme aminoglycoside-3′ adenyltransferase. Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90: 913-917. Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii.Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089.

Other selectable markers useful for plastid transformation are known inthe art and encompassed within the scope of the present disclosure.Typically, approximately 15-20 cell division cycles followingtransformation are required to reach a homoplastidic state. Plastidexpression, in which genes are inserted by homologous recombination intoall of the several thousand copies of the circular plastid genomepresent in each plant cell, takes advantage of the enormous copy numberadvantage, compared to nuclear genes, to achieve expression levels thatcan readily exceed 10% of the total soluble plant protein. Thus, incertain embodiments, a nucleotide sequence as disclosed herein isinserted into a plastid targeting vector and transformed into theplastid genome of a desired plant host. Plants homoplastic for plastidgenomes containing a nucleotide sequence of interest are obtained, andare capable of high-level expression of the nucleotide sequence.

Magnifection

Magnifection is a transient expression process that is based onexpression from viral RNA replicons delivered into plant cellssystemically using Agrobacterium. This method allows production ofrecombinant proteins at yields up to 5 g per kg of fresh leaf biomass,which approaches the biological limits for protein expression. Such highyields are possible because of the transient nature of the process,which allows the use of very potent amplicons derived from RNA virusessuch as Tobacco mosaic virus (TMV) or Potato virus X, without limitingbiomass accumulation, which takes place prior to infection. See, e.g.,Marillonnet et al. (2005) Nature Biotechnol. 23(6):718-723.

Additional disclosure of methods and compositions for plant geneitcengineering is provided in Bircher, J A (ed.) “Plant ChromosomeEnginerering: Methods and protocols.” Methods in Molecular Biology,vol.701, Springer Science+Business Media, 2011.

Computerized Systems and Media

Disclosed herein are computer readable media comprising the sequenceinformation of any of the nucleic acids disclosed herein; i.e., any ofSEQ ID NOs: 1-4533, any of the nucleic acids of embodiments 1-7, 15-17and 49-52, and any of the vectors of embodiments 8 and 9. In addition,the present disclosure includes computer-readable media comprising theamino acid sequence information of any of the polypeptides disclosedherein; i.e., any of SEQ ID NOs: 4534-8960 and any of the polypeptidesof embodiments 10-14 and 53. Such media include magnetic, optical,digital, electrical and hybrid media.

Also provided are computerized systems and computer program productscontaining the nucleic acids and polypeptide sequences disclosed hereinon a computer-readable medium. The computer systems can be local systemsinvolving a single computer connected to a database of the sequencesdisclosed herein, intranet systems, or systems including externalcomputers connected via the Internet. Such systems are used, forexample, to facilitate comparisons of the sequences disclosed hereinwith other known or unknown sequences.

Thus, a variety of computer systems designed to facilitate analysesusing the disclosed sequences are provided. Some systems include amemory, a system bus, and a processor. In certain embodiments, theprocessor is operatively disposed to: (i) compare one or more nucleotidesequences as disclosed herein with one or more second nucleotidesequences; (ii) identify identical or homologous sequences; and (iii)display the identified nucleotide sequence(s).

In additional embodiments, the processor is operatively disposed to: (i)compare one or more polypeptide sequences as disclosed herein with oneor more second polypeptide sequences; (ii) identify identical orhomologous sequences; and (iii) display the identified polypeptidesequence(s).

Also provided are computer systems that generally include a database anda user interface. The database in such systems comprises sequencerecords that include an identifier that identifies one or more projectsto which each of the nucleotide or amino acid sequence records belong.The user interface permits a user to input identifying informationspecifying which of the nucleotide or amino acid sequences are to becompared. It is also is also capable of displaying the identifiedpolynucleotide(s) or polypeptide(s).

Still other computer systems include a memory, a system bus, and aprocessor. The processor in such systems is operatively disposed to: (i)compare one or more polynucleotide sequences as disclosed herein withone or more known sequences to assess sequence similarity between one ormore of the polynucleotide sequences as disclosed herein and the one ormore known sequences; and (ii) display information concerning thesequence similarity between the one or more of the polynucleotidesequences disclosed herein and the one or more known sequences.

In additional embodiments, computer systems include a memory, a systembus, and a processor. The processor in such systems is operativelydisposed to: (i) compare one or more polypeptide sequences as disclosedherein with one or more known sequences to assess sequence similaritybetween one or more of the polypeptide sequences as disclosed herein andthe one or more known sequences; and (ii) display information concerningthe sequence similarity between the one or more of the polypeptidesequences disclosed herein and the one or more known sequences.

In addition to the various computer systems for conducting analyses andcomparisons, also provided are various computer program products forconducting the analyses and comparisons. Certain of the computer programproducts include program instructions for analyzing polynucleotidesequences by performing the following: (a) providing or receiving one ormore of the nucleotide sequences disclosed herein; (b) providing orreceiving a second nucleotide sequence; (c) determining the degree ofhomology or identity between the first nucleotide sequence and thesecond nucleotide sequence; and (d) displaying information concerningthe degree of homology or identity between the two nucleotide sequences.

In additional embodiments, computer program products include programinstructions for analyzing polypeptide sequences by performing thefollowing: (a) providing or receiving one or more of the amino acidsequences disclosed herein; (b) providing or receiving a second aminoacid sequence; (c) determining the degree of homology or identitybetween the first amino acid sequence and the second amino acidsequence; and (d) displaying information concerning the degree ofhomology or identity between the two amino acid sequences.

Also provided is a computer program product comprising acomputer-useable medium and computer-readable program code encodedwithin the computer-useable medium, wherein the computer-readableprogram code comprises (a) a database comprising the nucleotidesequences disclosed herein; and (b) effects the following steps with acomputer system (i) determining sequence similarity between one or morefirst nucleotide sequences as disclosed herein as compared to one ormore second sequences; and (ii) displaying the sequence similaritybetween the first and second nucleotide sequences. Furthermore, in anythese embodiments, the computer product can include or be operablylinked to a user interface, for example to query the database, displayinformation, etc.

Also provided is a computer program product comprising acomputer-useable medium and computer-readable program code encodedwithin the computer-useable medium, wherein the computer-readableprogram code comprises (a) a database comprising the amino acidsequences disclosed herein; and (b) effects the following steps with acomputer system (i) determining sequence similarity between one or morefirst amino acid sequences as disclosed herein as compared to one ormore second amino acid sequences; and (ii) displaying the sequencesimilarity between the first and second amino acid sequences.Furthermore, in any these embodiments, the computer product can includeor be operably linked to a user interface, for example to query thedatabase, display information, etc.

Additional disclosure of computer systems and computer-readable storagemedia are provided in U.S. Pat. No. 6,528,289, the disclosure of whichis incorporated by reference for the purpose of describing exemplarycomputer systems and computer-readable media.

Plant Growth Promotion

The compositions disclosed herein, in particular, C. subtsugae nucleicacids and polypeptides, can be used to modulate or more particularlypromote growth of plants, e.g. crops such as fruit (e.g., strawberry),vegetables (e.g., tomato, squash, pepper, eggplant), grain crops (e.g.,soy, wheat, rice, corn), trees, flowers, ornamental plants, shrubs(e.g., cotton, roses), bulb plants (e.g., onion, garlic) vines (e.g.,grape vine), and turf (e.g. bermudagrass, Kentucky bluegrass, fescues).The compositions can also be used to modulate the germination of aseed(s) in a plant(s).

C. subtsugae nucleic acids and polypeptides, or a formulated productthereof, can be used alone or in combination with one or more othercomponents as described below, such as growth promoting agents and/oranti-phytopathogenic agents in a tank mix or in a program (sequentialapplication called rotation) with predetermined order and applicationinterval during the growing season. When used in a combination with theabove-mentioned products, at a concentration lower than recommended onthe product label, the combined efficacy of the two or more products(one of which is the said composition disclosed herein) is, in certainembodiments, greater than the sum of each individual component's effect.Hence, the effect is enhanced by synergism between these two (or more)products, and the risk for the development of pesticide resistance amongthe plant pathogenic strains is reduced.

The composition can be applied by root dip at transplanting,specifically by treating a fruit or vegetable with the composition bydipping roots of the fruit or vegetable in a suspension of saidcomposition (about 0.25 to about 1.5% and more particularly about 0.5%to about 1.0% by volume) prior to transplanting the fruit or vegetableinto the soil.

Alternatively, the composition can be applied by drip or otherirrigation system. Specifically, the composition can be injected into adrip irrigation system. In a particular embodiment, the composition isapplied in a solution having a concentration of 1×10⁸ CFU /mL at a rateof about 11 to about 4 quarts per acre.

In yet another embodiment, the composition can be added as an in-furrowapplication. Specifically, the composition can be added as an in-furrowspray at planting using nozzles calibrated to deliver a total output of2-6 gallons/acre. Nozzles can be placed in the furrow opener on theplanter so that the pesticide application and seed drop into the furroware simultaneous.

Mixtures of the disclosed compositions with, for example, a solid orliquid adjuvant are prepared as known in the art. For example, mixturescan be prepared by homogeneously mixing and/or grinding the activeingredients with extenders such as solvents, solid carriers and, whereappropriate, surface-active compounds (surfactants). The compositionscan also contain additional ingredients such as stabilizers, viscosityregulators, binders, adjuvants as well as fertilizers or other activeingredients in order to obtain additional desired effects.

Combinations with Plant Growth Promoting Agents

The compositions disclosed herein can be used in combination with othergrowth promoting agents such as synthetic or organic fertilizers (e.g.,di-ammonium phosphate, in either granular or liquid form), compost teas,seaweed extracts, plant growth hormones such as IAA (indole acetic acid)used in a rooting hormone treatment for transplants either alone or incombination with plant growth regulators such as IBA (indole butyricacid) and NAA (naphthalene acetic acid), and growth promoting microbes,such as, for example, methylotrophs, PPFM (Pink Pigmented FacultativeMethylotrphs), Bacillus spp., Pseudomonads, Rhizobia, and Trichoderma.

Seed Coating Agents

The compositions disclosed herein can also be used in combination withseed-coating agents. Such seed coating agents include, but are notlimited to, ethylene glycol, polyethylene glycol, chitosan,carboxymethyl chitosan, peat moss, resins and waxes or chemicalfungicides or bactericides with either single site, multisite or unknownmode of action.

Anti-Phytopathogenic Agents

The compositions disclosed herein can also be used in combination withother anti-phytopathogenic agents, such as plant extracts,biopesticides, inorganic crop protectants (such as copper), surfactants(such as rhamnolipids; Gandhi et al., 2007) or natural oils such asparaffin oil and tea tree oil possessing pesticidal properties orchemical fungicides or bactericides with either single site, multisiteor unknown mode of action. As defined herein, an “anti-phytopathogenicagent” is an agent that modulates the growth of a plant pathogen,particularly a pathogen causing soil-borne disease on a plant, oralternatively prevents infection of a plant by a plant pathogen. Plantpathogens include but are not limited to fungi, bacteria, actinomycetesand viruses.

An anti-phytopathogenic agent can be a single-site anti-fungal agentwhich can include but is not limited to benzimidazole, a demethylationinhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole),morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate,quinone outside inhibitor, quinoline, dicarboximide, carboximide,phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon,cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine,phthalimide, benzenoid (xylylalanine). In a more particular embodiment,the antifungal agent is a demethylation inhibitor selected from thegroup consisting of imidazole, piperazine, pyrimidine and triazole(e.g., bitertanol, myclobutanil, penconazole, propiconazole,triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole,hexaconazole, tebuconazole, tetraconazole). In a most particularembodiment, the antifungal agent is myclobutanil. In yet anotherparticular embodiment, the antifungal agent is a quinone outsideinhibitor (e.g., strobilurin). The strobilurin may include but is notlimited to azoxystrobin, kresoxim-methyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether).

In yet a further embodiment, the fungicide is a multi-sitenon-inorganic, chemical fungicide selected from the group consisting ofchloronitrile, quinoxaline, sulphamide, phosphonate, phosphite,dithiocarbamate, chloralkythios, phenylpyridine-amine, andcyano-acetamide oxime.

In yet a further embodiment, the anti-phytopathogenic agent can bestreptomycin, tetracycline, oxytetracycline, copper, or kasugamycin.

Bioremediation

The C. subtsugae genome encodes genes involved in the metabolism of,inter alia, phosphorus, iron and aromatic compounds. See, e.g., Table 6supra. Such genes and their gene products can be used in bioremediationmethods. For instance, genes and sequences realted to metal tansport,metal accumulation, degradation of organic compounds, and othermetabolite transformation can be engineered into plants with the purposeof applying the transformed plant to bioremediation of soils, sediment,water, and other polluted substrates. Protocols for the transformationof Indian mustard (Brassica juncea), sunflower (Helianthus annus),tomato and yellow poplar (Liriodendron tulipifera) are known. See, e.g.,Eapen and D'Souza (2005); Mello-Farias and Chavez (2008).

Plants can be transformed with Cytochrome P450-encoding genes toincrease their resistance to particular pollutants, both organic andinorganic. Transformation with nucleic acids encoding enzymes involvedin gluthatione conjugation (for example, glutathione S-transferases) canincrease rates of xenobiotic detoxification. Plants expressing bacterialnitroreductases can be used for the detoxification of nitrate organiccompounds, such as explosives.

Uses of transgenic plants for phytoremediation applications has beendescribed, for example, by Abhilash et al. (2009); Van Aken et al.(2010); Doty (2008) and Macek et al. (2008).

EXAMPLES Example 1 Cell Growth and DNA Extraction

Chromobacterium subtsugae PRAA-1 was grown in 200 ml LB broth in 1Lflasks at 26° C. with rotation at 150 rpm for 24-48 hours. Biomass washarvested from the culture by centrifugation.

Genomic DNA was extracted using the MoBio Power Microbial Maxi-DNAExtraction Kit (MoBio Cat No. 122223-25). DNA was eluted in 1.5 ml ofelution buffer (included in kit). To assess DNA quality and quantity, a10 uL aliquot was loaded into a 1.5% agarose gel and electrophoresis wasconducted for 30 minutes at 100 V. DNA was visualized with a UVtransilluminator using EZ-Vision loading dye. Over 100 ug of DNA wererecovered.

Example 2 DNA Sequence Determination and Assembly

DNA sequences were determined using a HiSeq 2000 (Illumina, San Diego,Calif.), with sequence reads of 100 bp, pair ended, aiming for a minimumcoverage of 40×. Final data consisted of two sets of paired-end samplesin FASTQ format, providing approximately 200× coverage of the genome.

The four FASTAQ files were used for assembly. FASTAQ sequences weresubjected to quality control using FASTQC, and the average distancebetween pairs was calculated by comparing the first 10,000 pairs fromboth sets with the initial assembled contigs using BWA. Li & Durbin(2009) Bioinformatics 25(14):1754-1760. TrimGalore (BabrahamBioinformatics, Cambridge, UK) was then used to generate twohigh-quality paired-end sets and four single-read files for thosesequences whose partner read was below the quality threshold of at least50 nucleotides after clipping on Q2.

Sequence reads were assembled using Ray assembler v2.0.0. Boisvert etal. (2010) J Comput Biol. 17(11):1519-1533. A titration of kmer sizeswas performed with a kmer range of 19-63; resulting in successfulassemblies at 19, 21, 31, 41, 47, 49 and 63. Further scaffolding wasperformed using SSPACE v1.1 using all available reads on the scaffoldsproduced by the Ray analysis. Boetzer et al. (2011) Bioinformatics27(4):578-579. Gaps were connected using GapFiller, with a maximumiteration of twenty steps. Boetzer & Pirovano (2012) Genome Biol.13(6):R56. The resulting scaffolds were mapped against the referencegenome of Chromobacterium violaceum ATCC 12742, using CONTIGuator withan e value of 1e-10. Galardini et al. (2011) Source Code Biol. Med.6:11.

To confirm contig and scaffold orders, the alignments were inspectedmanually using ACT. Carver et al. (2008) Bioinformatics24(23):2672-2676. The original dataset was mapped back onto theChromobacterium subtsugae sequence using BWA (Li & Durbin, supra) with aseed length of 19.

This process yielded a high quality genome of 4,690,330 bases with atotal of 145,992 bases in contigs not matching the reference genome(Chromobacterium violaceum) and 4,264 undefined nucleotides (N's) in 42gaps. Subsequent filling of the gaps in pseudocontigs closed 8 of the 42gaps and extended the pseudocontigs to 4,704,820 bases where most gapsare single ‘N’ positions with only 2 gaps remaining in positions2,153,178-2,153,283 (105 bases) and 2,474,439-2,474,486 (47 bases).

Example 3 Genome Annotation

Initial predictions were obtained using RAST. Meyer et al. (2008) BMCBioinformatics 9:386. These predictions utilized pseudocontigs andcontigs that were rejected by CONTIGuator. The analysis yielded 4,467CDS predictions, 92 tRNA predictions, 26 rRNA genes and 91 putativemissing genes.

Example 4 General Features of the Chromobacterium substsugae Genome

The genome of Chromobacterium subtsugae is a circular DNA molecule of4,705,004 bp. No extrachromosomal plasmids were discovered during genomeanalysis.

Using RAST, 4532 features were identified, out of which 4415 were codingsequences, as well as 117 RNA sequences. Using RAST, it was possible toassign 1980 features to functional subsystems (about 45% of total), outof which 104 were hypothetical. Features not assigned to subsystemsaccounted for 55% of the total (2435 features) with 1280 beinghypothetical.

Comparison to the most closely related organism, Chromobacteriumviolaceum, indicated that Chromobacterium subtsugae posseses 174functional features that are not shared with Chromobacterium violaceum,181 features are present in C. violaceum that are not present in C.substugae, and both organisms had 2179 functional features in common. Incomparison with all sequences in C. violaceum, 3398 C. subtsugaesequences were found to have over 50% similarity, 2518 sequences hadmore than 80% similarity, and 1369 sequences had more than 90%similarity.

Example 5 Condon Usage in C. subtsugae

Codon usage bias is an important parameter in the optimization of theexpression of heterologous genes, and for regulating the expression ofgenes in a particular host. For example, a codon usage table can be usedto direct the modification of a nucleotide sequence so that it includescodons more preferabe to the host, yet encodes the same amino acidsequence, in order to maximize expression of one or more desiredproteins or peptides.

Based on SEQ ID NO: 1, a codon usage table for C. subtsugae wasgenerated using CUSP software(emboss.bioinformatics.nl/cgi-bin/emboss/cusp) and is shown in Table 15.

TABLE 15 C. subtsugae codon usage #CdsCount: 18257 #Coding GC 65.96%#1st letter GC 67.83% #2nd letter GC 62.69% #3rd letter GC 67.37% #CodonAA Fraction Frequency Number GCA A 0.146 20.669 28518 GCC A 0.332 46.93664761 GCG A 0.325 45.959 63412 GCT A 0.197 27.880 38467 TGC C 0.72619.161 26437 TGT C 0.274 7.235 9983 GAC D 0.598 22.148 30559 GAT D 0.40214.859 20502 GAA E 0.547 16.860 23263 GAG E 0.453 13.952 19250 TTC F0.725 15.255 21048 TTT F 0.275 5.772 7964 GGA G 0.164 15.235 21020 GGC G0.555 51.502 71061 GGG G 0.135 12.517 17270 GGT G 0.146 13.541 18684 CACH 0.493 13.794 19033 CAT H 0.507 14.169 19550 ATA I 0.122 3.317 4577 ATCI 0.679 18.451 25458 ATT I 0.199 5.409 7463 AAA K 0.345 8.220 11341 AAGK 0.655 15.603 21528 CTA L 0.080 5.873 8103 CTC L 0.100 7.354 10147 CTGL 0.578 42.678 58886 CTT L 0.113 8.372 11551 TTA L 0.026 1.884 2599 TTGL 0.104 7.638 10539 ATG M 1.000 13.457 18568 AAC N 0.620 11.782 16257AAT N 0.380 7.219 9960 CCA P 0.222 18.360 25332 CCC P 0.157 12.928 17837CCG P 0.444 36.649 50567 CCT P 0.177 14.631 20187 CAA Q 0.449 18.18125085 CAG Q 0.551 22.345 30831 AGA R 0.048 6.454 8905 AGG R 0.080 10.82514936 CGA R 0.167 22.617 31206 CGC R 0.383 51.783 71448 CGG R 0.23732.054 44227 CGT R 0.086 11.595 15998 AGC S 0.305 20.807 28709 AGT S0.062 4.216 5817 TCA S 0.122 8.315 11473 TCC S 0.182 12.443 17168 TCG S0.242 16.564 22855 TCT S 0.087 5.977 8247 ACA T 0.158 7.185 9914 ACC T0.405 18.418 25413 ACG T 0.323 14.686 20263 ACT T 0.114 5.166 7128 GTA V0.076 3.252 4487 GTC V 0.291 12.486 17228 GTG V 0.453 19.419 26793 GTT V0.180 7.709 10637 TGG W 1.000 24.015 33135 TAC Y 0.663 8.606 11874 TAT Y0.337 4.380 6043 TAA * 0.086 1.143 1577 TAG * 0.112 1.481 2043 TGA *0.802 10.608 14637

Example 6 Identification of Gene Clusters Related to PolyketideSynthesis and Other Secondary Metabolite Production

Secondary metabolite production clusters were examined using the antiSMASH program (antismash.secondarymetabolites.org/). As shown in Table16, several putative clusters were identified, as well as four NRPSclusters, one indole cluster, one terpenoid cluster, one bacteriocincluster, and one butyrolactone cluster. The amino acid compositions ofNRPS sequences were predicted using NRSPredictor2(nrps.informatik.uni-tuebingen.de).

TABLE 16 Cluster Type From To Cluster 1 Putative 129943 134127 Cluster 2Putative 290722 315490 Cluster 3 Putative 323716 329226 Cluster 4Putative 371894 394333 Cluster 5 Putative 885815 893212 Cluster 6 Nrps1566319 1628592 Cluster 7 Putative 2210421 2228951 Cluster 8 Nrps2299432 2347915 Cluster 9 Putative 2352275 2367119 Cluster 10 Putative2384147 2393105 Cluster 11 Nrps-t1pks 2424775 2490818 Cluster 12Bacteriocin 2890220 2901104 Cluster 13 Putative 2949745 2965040 Cluster14 Putative 3074586 3081909 Cluster 15 Terpene 3170248 3191973 Cluster16 Indole 3534153 3557149 Cluster 17 Putative 3667563 3693003 Cluster 18Bacteriocin 3801030 3811854 Cluster 19 Putative 4148365 4165333 Cluster20 Butyrolactone 4208155 4218943 Cluster 21 Putative 4254490 4291018Cluster 22 Nrps 4337664 4385597

Example 7 Construction of a Cosmid Library from Chromobacteriumsubtsugae PRAA-1

A cosmid library is constructed to screen for C. subtsugae genes withnovel activities relating to agriculture, pest control, pharmaceuticalapplication, etc. Genomic DNA is isolated from Chromobacterium subtsugaeby growing the isolate in suitable liquid media, for example LB,nutrient broth, or YM broth. Genomic DNA is extracted and purified usinga commercial kit, such as PureLink Genomic DNA (Life Technologies),UltraClean DNA extraction Kit (MoBio), or Quiagen DNEasy kits.Alternatively, freshly grown cells are pelleted by centrifugation andresuspended in TE buffer (100 mM Tris pH 8, 10mM EDTA) with 2 mg/mllysozyme for 30 minutes at 37° C. The suspension is then treated withProteinase K and SDS to remove protein and lipids (100 ug/ml ProteinaseK in 1% SDS, 50 mM EDTA and 1M urea) and incubated 55° C. for 10minutes. Following extraction with phenol-chloroform-isoamyl alchohol(25:24:1), the aqueous phase is recovered and mixed with 0.6 volumes ofisopropanol (molecular grade) to precipitate the DNA. DNA is pelleted bycentrifugation, washed with 70% ethanol at least twice, and the cleanpellet is dried and resuspended in 0.5× TE buffer.

The clean DNA is digested with Sau3AI (New England Biolabs, Beverly,Mass.), using 0.5 units of enzyme per ug of DNA at 37° C., in 100 ul ofbuffer according to the manufacturer's recommendations. The digestionreaction is sampled at regular time intervals to determine a time pointthat provides fragments in the 40 kb range.

The library is prepared using a commercially available vector ligationkit such as SuperCos1 Cosmid Vector Kit (Agilent Technologies) followingthe manufacturer's durections. The ligation mixture is into phage usinga commercially availble kit, such as Gigapack XL III (AgilentTechnologies), following the manufacturer's directions. Phage are usedto infect competent cells such as E. coli XL-1MR (Agilent Technologies).

The cosmid library is plated on LB agar or other suitable media,supplemented with 50 ug/ml kanamycin. Inoculated plates are incubatedovernight (up to 18 hours) at 37° C. At least 1000 colonies are pickedfrom the plates and transferred to duplicate 96-well plates loaded withLB or other suitable liquid media. Multi-well plates are incubatedovernight with agitation. One set of plates is used for screening, andthe duplicate is stored at −80° C. after addition of 25% glycerol.

Example 8 Screening of a Cosmid Library for Clones Encoding LepidopteranInsecticide Activity

Cosmid-containing cells are grown overnight in 96-well plates and areassayed using a diet-overlay method in which a sample of cells, cellbroth, cell supernatant or cell extract is deposited on the surface of adiet-loaded 96-well plate and allowed to dry. Lepidopteran eggs,neonates or larvae of target insect (e.g., Heliothis virescens,Trichlopusia ni, Spodoptera exigua, Plutella xylostella, Manduca sexta,etc.) are loaded into each well, and the plates are incubated for 5 to 7days. Each well is then evaluate d for hatching, mortality, stunting,and lack of food consumption. Cosmid clones with insectidal activity(e.g., death, lack of hatching, reduced feeding) are identified.

Example 9 Screening of a Cosmid Library for Clones Encoding NematicideActivity

Cosmid-containing cells are grown overnight in 96-well plates andassayed using a 96-well motility test in which cells, cell broth, cellsupernatant or cell extract is deposited into the wells, and freshlyhatched nematode juveniles (J2s) are then introduced into the wells(e.g., Meloidogyne hapla, Meloidogyne incognita, Globodera sp.,Heterodera sp. etc.). Following addition of nematodes, the plates areincubated for 2 to 5 days, and each well is then evaluated for nematodemotility. Paralyzed or dead nematodes appear straight while livenematodes move and have a cuved or curled shape. Extracts, cells,supernatant and/or broth from clones with nematicide activity areidentified.

The assay can be modified to evaluate nematode egg hatching. In thiscase, the screening plates are loaded with the test substance (cells,cell broth, cell supernatant or cell extract), and then a known numberof nematode eggs are added. Hatching is measured by counting juvenilesafter 2-3 days of incubation and comparing to an untreated control.Extracts, cells, supernatant and/or broth from clones that inhibitnematode egg hatching are identified.

Example 10 Screening of Cosmid Library for Clones Encoding AlgaecideActivity

Cosmid-containing cells are grown overnight in 96-well plates. Targetalgae (e.g., Chlamydomonas reinhardtii, Pseudokirchenella subcapitata,Spyrogyra sp., Microcystis aurantiaca, Anabaena sp., etc.) are grown inErlenmeyer flasks under lights, and dispensed into 96-well plates. Thetest substance (cells, supernatants, whole cell broth or extracts) isdeposited into the wells, optionally with the use of a robot. Loadedplates are incubated for 3 days under lights. Algaecide activity isevident by decrease in chlorophyll production. Plates can be scoredvisually, or by measuring chlorophyll fluorescence using a multi-wellUV-visible spectrophotometer.

Example 11 Screening of Cosmid Library for Acaricide Activity

Cosmid-containing cells are grown overnight in multiple 96-well platesto obtain the desired amount of test substance. The acaricide bioassayis performed on excised leaf disks that are treated with the cells; orwith extracts, supernatant, or whole cell broth derived therefrom. Smallexcised plant leaves or leaf disks are treated by applying the testsubstance to the surface. After the test substance has dried, targetpests are introduced onto the leaf and mortality is evaluated after apredestined period of time.

The type of plant used for the assay is selected according to the targetpest. For instance, for two-spotted spider mite (T. urticae), femaleadults (from a synchronized colony) are introduced to excised kidneybean leaf that has been treated with the test solution. Mortality isdetermined 2-3 days after treatment.

For western flower thrips (F. occidentalis), 10-12 first instar larvaeare introduced onto an excised kidney bean leaf that has been treatedwith the test substance, and mortality is evaluated after 2-3 days.

Example 12 Characterizations of Active Clones Obtained from FunctionalScreens

DNA is extracted from cosmid clones expressing activity in any of thescreening assays described in examples 8-11, or in any other functionalscreening assay. DNA can be isolated with the use of a commercial kit(e.g., MoBio UltraClean, Qiagen DNAEasy, etc.) or by alkaline lysis asdescribed by Maniatis et al. (1989). Restriction enzyme digestion andgel electrophoresis can be used to compare the DNA content of clones.

DNA fragments of interest are subcloned using art-recognized methods,optionally with the use of a commercial kit, e.g., pGEM-T Vector System(Promega, Madison, Wis.) and expressed, e.g., in E. coli. The subclonescan be re-screened in the functional bioassay and the DNA fragment(s)associated with the detected activity (e.g., toxin production) can beidentified.

Identified DNA fragment(s) can be sequenced and mapped on the C.subtsugae genome, and can be used for the design of probes, e.g., forscreening the genomes of C. subtsugae and other organisms for toxinbiosynthetic genes. Fragments identified in this way can also beexpressed in a heterologous host, or used to transform a plant.

Example 13 Transformation of Tomato (Solanum lycopersicum) withAgrobacterium

The following procedure is adapted from Sharma, M. K. et al. 2009.“Asimple and efficient Agrobacterium-mediated procedure for transformationof tomato.” Journal of Biosciences 34:423-433.

Media and Solutions

The composition of various media is described in Table 17. Mediacomponents, except agar, are combined according to Table 17 and adjustedto pH 5.8 using 1N KOH, before adding plant-tissue culture grade agar.Stock solutions of BAP (6-benzylmaino purine) and zeatin are prepared indimethyl sulphoxide (DMSO). Antibiotic stock solutions are prepared indeionized water and filter-sterilized. Agrobacterium strain AGL1 isgrown on YEM agar or broth containing 100 mg/l rifampicin and 50 mg/lkanamycin.

Preparation of Agrobacterium

Agrobacterium tumefaciens, transformed with the gene or genes ofinterest, (e.g., any of the genes disclosed in any of Tables 2-13) isgrown in YEM medium with rifampicin and kanamycin, in shaking culturefor 72 h at 28° C. and 200 rpm. Cells are pelleted by centrifugation,washed and resuspended in WS medium. Bacterial density is determined bymeasuring OD₆₀₀ and the final cell concentration is adjusted to ˜10⁸cells/ml by diluting with WS medium.

Plant Transformation

Middle pieces (0.7×1.0 cm) from 10-day cotyledons are collected byexcising at the tip and base. The sections are pre-cultured for 48 hoursat 28° C. on M1 medium, with the adaxias surface in direct contact withthe medium.

Healthy explants are selected and incubated in Agrobacterium suspensionfor 30 minutes, with inversion every 10 minutes. Explants are blotted onsterile tissue paper and returned to M1 agar (50-80 explants per plate)for an additional 72 hours. The explants are then washed 4-5 times in WSmedium, blotted on sterile tissue paper and transferred to SM containing1 mg/L trans-zeatin for regeneration (20-25 explants per regenerationplate).

Regeneration plates are incubated at 28° C. under a 16/8 light/darkcycle. Regeneration is evidenced by development of a callus. Regeneratedexplants are selected and transferred to fresh SM medium every 15 days.

Regenerated shoots can be excised from the callus and transferred to RMmedium.

Plantlets that are at least 2 inches in height and have strong roots areselected for transfer to pots. Planting substrate consists of pottingsoil mixed 1:1 with 1:1:1 vermiculite: perlite: sphagnum.

TABLE 17 M1 M2 WS SM RM MS Salts 0.5x 1x 1x 1x 1x (Murashige and Skoog,1962) Gamborg's B5 vitamins 0.5x 1x 1x 1x 1x Sucrose (g/L) 15 30 30 3030 Agar (% w/v) 0.8 0.8 0 0.8 0.8 BAP (mg/L) 0 2 0 0 0 Kanamycin (mg/L)0 0 0 100 100 Cefotaxime (mg/L) 0 0 0 500 500

Example 14 Creation of Transgenic Soybean Plants Comprising anInsecticidal Gene from Chromobacterium substugae

Mature glycine max seeds are surface sterilized with chlorine gas insidea bell jar under a fume hood. Seeds are kept in 100×20 mm Petri disheswith chlorine gas produced by pouring 100 ml of 4% sodium hypochloriteinto a beaker and adding 5 ml of 12N hydrochloric acid. Aftersterilization, seeds are placed on germination medium (GM; MS basalsalts with vitamins, 3% sucrose, 0.8% plant agar, and 1 mg/L BAP,optimized from regeneration experiment, pH 5.8). Murashige and Skoog,1962. Seeds are germinated under fluorescent light or darkness at 24±1°C. for 5-7 days to compare transformation frequency.

The method described here is a modification of that described by Zhanget al. (1999) Plant Cell, Tissue and Organ Culture 56:37-46. Twocotyledonary explants are obtained by cutting a horizontal slice throughthe hypocotyl with a No. 11 surgical blade. The hypocotyl issubsequently removed and ten scratches are made at the surface ofcotyledonary node regions. Explants are immersed for 30 min in asuspension of A. tumefaciens which has been engineered to comprise thegene of interest, e.g., a gene that encodes an insecticidal protein, ora protein that is involved in the synthesis of an insecticidal compound.See Tables 2-13 above for listings of exemplary genes of interest.Following immersion, ten explants are randomly placed on sterile filterpaper placed on solid co-cultivation medium (CM; Gamborg's B5 basalsalts with vitamins, 3% sucrose, 20 mM MES, 3.3 mM L-cysteine, 1 mMdithiothreitol, 0.1 mM acetosyringone, 0.8% plant agar, pH 5.4) (Gamborget al., 1968) in 100×20 mm Petri dishes, and incubated at 24±1° C. for 5days under dark conditions.

After 5 days of co-cultivation , explants are briefly washed in liquidshoot induction medium (SIM; Gamborg's B5 basal salts with vitamins, 3%sucrose, 3 mM MES, 1.67 mg/L BAP, 250 mg/L cefotaxime, pH 5.7) to removeexcess A. tumefaciens on explants. Explants are then transferred tosolidified SIM without PPT to stimulate shoot induction for the first 14days, after which the explants are sub-cultured on fresh SIM containing5 mg/L PPT for selection of transformed shoots. Organogenic shoots fromthe explants are trimmed and then transferred to shoot elongation medium(SEM; MS basal salts with vitamins, 3% sucrose, 3 mM MES, 0.5 mg/Lgiberellic acid, 50 mg/L asparagine, 1 mg/L zeatin, 0.1 mg/Lindole-3-acetic acid, 250 mg/L cefotaxime, 50 mg/L vancomycin, 0.8%plant agar, 5 mg/L PPT, pH 5.7). Explants are transferred to new SEMmedium every 14 days, and surviving shoots are planted on root inductionmedium (RIM; MS basal salts with vitamins, 3% sucrose, 1 mg/Lnaphthalene acetic acid, 0.8% plant agar, pH 5.7) and grown until rootsdevelop. After acclimation, the transgenic plants are transplanted topotting soil and maintained in a greenhouse. Selection is carried out byPCR. See also Lee, et al. (2011) J. of Korean Soc. Appl. Biol. Chem. 54:37-45.

Example 15 Efficacy of two identified proteins against Corn rootworm(Diabrotica undecimpunctata) CRW

SEQ ID NO:8924 and SEQ ID NO:7904 proteins were enriched and partiallyresolved from each other using strong cation and strong anion exchangeresins and by hydrophobic interaction chromatography. Proteinconcentration was estimated using the Invitrogen Quant-iT assaycalibrated with BSA. Proteins were buffered to approximately pH 6 with20 mM MES or pH 7.5 with tris-HCl and were adjusted to 1 mg/mL totalprotein prior to bioassay.

Proteins were matched to their amino acid sequences by peptide spectrummatching. Excised protein bands were digested into peptides with trypsinand analyzed by LC-MS using an Agilent 6540 mass spectrometer. Recordedspectra were matched using the x!Tandem, PeptideProphet, andProteinProphet software packages.

Activity against Corn rootworm was tested on Diet Overlay Bioassays. Theappropriate artificial insect diet was dispensed into each well of astandard 96 well plate and allowed to dry. Once the diet solidified, 100uL of the treatment was pipetted into the appropriate number of wellsand allowed to dry. A single 1st instar larva was delivered into eachwell of a 96 well plate. Mortality was scored at 3 days after treatment.

Two proteins (SEQ ID NO:8924 and SEQ ID NO:7904) were tested induplicates (Exp1 and Exp2) for insecticidal activity against Cornrootworm (Diabrotica undecimpunctata) CRW. Mortality was scored 3 dayspost treatment in two independent experiments. Results are shown inTable 18.

TABLE 18 % Mortality Summary Exp1 Exp2 SEQ ID  90 58.33 NO: 8924 SEQ ID100 33.33 NO: 7904

The inventions described and claimed herein are not to be limited inscope by the specific aspects herein disclosed, since these aspects areintended to be illustrative. Any equivalent aspects are intended to bewithin the scope of the disclosure. Indeed, various modifications of themethods and compositions shown and described herein will be apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

1-12. (canceled)
 13. A cell comprising: a recombinant vector having aheterologous promoter operably linked to a nucleic acid encoding apolypeptide with 95% identity to SEQ ID NO:
 8924. 14. A plant, a plantpart, or a seed comprising: one or more cells comprising a recombinantvector comprising a heterologous promoter operably linked to a nucleicacid encoding a polypeptide with 95% identity to SEQ ID NO:8924.
 15. Theplant part of claim 2, wherein said plant part is selected from thegroup consisting of pollen, ovule, flower, shoot, root, stalk, silk,tassel, ear, and leaf tissue.
 16. The cell of claim 1, wherein said cellis a bacterial, mammalian, or fungal cell.
 17. A method of producing aninsect resistant plant cell, said method comprising the step of:transforming a recombinant vector comprising a heterologous promoteroperably linked to a nucleic acid encoding a polypeptide with 95%identity to SEQ ID NO:8924, into a plant cell.
 18. An anti-counterfeitmilled seed comprising: a plant cell comprising a recombinant vectorhaving a heterologous promoter operably linked to a nucleic acidencoding a polypeptide with 95% identity to SEQ ID NO: 8924, wherein thepolypeptide provides an indication of plant cell origin.
 19. Apesticidal composition comprising: an isolated and purified polypeptidehaving the sequence with 95% identity to SEQ ID NO:8924, and one or moreartificial pesticides disposed in a carrier.
 20. The pesticidalcomposition of claim 7, wherein at least one of the one or moreartificial pesticides composition is an insecticide.
 21. A method formodulating a pest infestation in a plant, said method comprising thestep of: contacting a plant or a plant part with an amount of apesticidal composition comprising (a) a polypeptide having the sequencewith 95% identity to SEQ ID NO: 8924 and (b) one or more artificialpesticides dispose in a carrier, said amount effective to modulate saidpest infestation.
 22. The method of claim 9, wherein the pest isselected from the group consisting of insects, fungi, nematodes,bacteria and mites.
 23. The method of claim 10, wherein the insectscomprise cabbage loopers, lygus, beet armyworms, corn rootworm, ordiamondback moth.
 24. A seed or seed coating composition comprising: apolypeptide with 95% identity to SEQ ID NO: 8924, and one or moreartificial pesticides disposed in a carrier.